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"schema_version": "seqcolyte.spec.v1",
"spec_id": "cel_seq",
"assay": "CEL-Seq",
"chemistry_version": "",
"platform": "illumina",
"platform_params": {
"read_type": "short"
},
"source_docs": [
{
"doc_id": "CEL-Seq_protocol.pdf",
"title": "CEL-Seq_protocol.pdf",
"url": null,
"path": "/Users/seqmachines/playground/protocols-test/protocols/cel_seq/CEL-Seq_protocol.pdf",
"retrieved_date": null
},
{
"doc_id": "CEL-Seq_paper.pdf",
"title": "CEL-Seq_paper.pdf",
"url": null,
"path": "/Users/seqmachines/playground/protocols-test/protocols/cel_seq/CEL-Seq_paper.pdf",
"retrieved_date": null
}
],
"oligos": [
{
"oligo_id": "celseq_rt_primer",
"name": "CEL-Seq barcoded RT primer",
"aliases": [],
"role": "Reverse-transcription / capture primer carrying anchored poly(dT), cell barcode, 5' Illumina small-RNA adapter and T7 promoter",
"kind": "assembled",
"sequence": "CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATC[CELL_BARCODE:8]TTTTTTTTTTTTTTTTTTTTTTTTV",
"direction": "5_to_3",
"components": [
{
"name": "5' leader",
"sequence": "CGATTGAGGCCGG",
"role": "5' linker/leader upstream of T7 promoter"
},
{
"name": "T7 promoter",
"sequence": "TAATACGACTCACTATAGGG",
"role": "T7 RNA polymerase promoter (drives IVT), as in Baugh et al. 2001"
},
{
"name": "Illumina 5' RNA adapter (RA5)",
"sequence": "GTTCAGAGTTCTACAGTCCGACGATC",
"role": "5' Illumina small-RNA sequencing adapter"
},
{
"name": "Cell barcode",
"sequence": "[CELL_BARCODE:8]",
"role": "8-bp cell/sample barcode read at the start of read 1"
},
{
"name": "Anchored poly(dT)",
"sequence": "TTTTTTTTTTTTTTTTTTTTTTTTV",
"role": "24-nt poly(dT) with 3' V anchor for polyA mRNA capture"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Working conc. 25 ng/ul, desalted. Barcodes are 8 bp, designed in groups of four so the first five positions have equal base representation; each pair differs by >=2 nt. The 40 published barcodes (inserted at [CELL_BARCODE:8], 5'->3'): 1 CATCACGC, 2 GTCGTCGC, 3 ACGACCGC, 4 TGATGCGC, 5 CATCAATC, 6 GTCGTATC, 7 ACGACATC, 8 TGATGATC, 9 CATCATCC, 10 GTCGTTCC, 11 ACGACTCC, 12 TGATGTCC, 13 CATCAGAA, 14 GTCGTGAA, 15 ACGACGAA, 16 TGATGGAA, 17 TCACACGC, 18 CGTGTCGC, 19 GACACCGC, 20 ATGTGCGC, 21 TCACATCA, 22 CGTGTTCA, 23 GACACTCA, 24 ATGTGTCA, 25 TCACAGAG, 26 CGTGTGAG, 27 GACACGAG, 28 ATGTGGAG, 29 CTAACCGC, 30 GCTTGCGC, 31 AGCCACGC, 32 TAGGTCGC, 33 CTAACTCA, 34 GCTTGTCA, 35 AGCCATCA, 36 TAGGTTCA, 37 CTAACGAG, 38 GCTTGGAG, 39 AGCCAGAG, 40 TAGGTGAG."
},
{
"oligo_id": "illumina_5p_rna_adapter_ra5",
"name": "Illumina 5' RNA adapter (RA5)",
"aliases": [],
"role": "5' Illumina small-RNA adapter; supplied within the CEL-Seq RT primer",
"kind": "single",
"sequence": "GTTCAGAGTTCTACAGTCCGACGATC",
"direction": "5_to_3",
"components": [],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "5' Illumina adapter 'as used in the Illumina small RNA kit'; this segment is embedded in the CEL-Seq RT primer, so its sequence is documented directly."
},
{
"oligo_id": "illumina_3p_rna_adapter_ra3",
"name": "Illumina 3' RNA adapter (RA3)",
"aliases": [],
"role": "3' Illumina small-RNA adapter ligated to fragmented aRNA",
"kind": "single",
"sequence": "TGGAATTCTCGGGTGCCAAGG",
"direction": "5_to_3",
"components": [],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "TruSeq Small RNA RA3 adapter; protocol uses it diluted 1:5. Sequence cited in the protocol as 'available from Illumina' and not printed there; shown is the standard Illumina TruSeq Small RNA RA3 sequence."
},
{
"oligo_id": "rna_rt_primer_rtp",
"name": "RNA RT Primer (RTP)",
"aliases": [],
"role": "Reverse-transcription primer complementary to the ligated 3' adapter (RA3)",
"kind": "single",
"sequence": "GCCTTGGCACCCGAGAATTCCA",
"direction": "5_to_3",
"components": [],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "From the Illumina TruSeq Small RNA kit; primes SuperScript II RT of the RA3-ligated RNA. Sequence not printed in the protocol ('available from Illumina'); shown is the standard TruSeq Small RNA RTP."
},
{
"oligo_id": "rna_pcr_primer_rp1",
"name": "RNA PCR Primer (RP1)",
"aliases": [],
"role": "Forward library PCR primer (adds P5 and completes the 5' adapter)",
"kind": "assembled",
"sequence": "AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA",
"direction": "5_to_3",
"components": [
{
"name": "P5",
"sequence": "AATGATACGGCGACCACCGAGATCTACAC",
"role": "Illumina P5 flow-cell adapter"
},
{
"name": "Partial RA5",
"sequence": "GTTCAGAGTTCTACAGTCCGA",
"role": "5' small-RNA adapter overlap"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "From the Illumina TruSeq Small RNA kit. Sequence not printed in the protocol ('available from Illumina'); shown is the standard TruSeq Small RNA RP1."
},
{
"oligo_id": "indexed_rna_pcr_primer_rpix",
"name": "Indexed RNA PCR Primer (RPIX)",
"aliases": [],
"role": "Reverse indexed library PCR primer (adds i7 sample index and P7)",
"kind": "assembled",
"sequence": "CAAGCAGAAGACGGCATACGAGAT[SAMPLE_INDEX:6]GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA",
"direction": "5_to_3",
"components": [
{
"name": "P7",
"sequence": "CAAGCAGAAGACGGCATACGAGAT",
"role": "Illumina P7 flow-cell adapter"
},
{
"name": "i7 sample index",
"sequence": "[SAMPLE_INDEX:6]",
"role": "6-nt Illumina library index (read as separate index read)"
},
{
"name": "RA3/RTP overlap region",
"sequence": "GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA",
"role": "3' adapter overlap, complementary to RA3"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "RPIX = uniquely indexed reverse PCR primer from the Illumina TruSeq Small RNA kit; provides an additional per-library barcode so multiple IVT pools run together. Sequence not printed in the protocol ('available from Illumina'); shown is the standard TruSeq Small RNA RPI scaffold."
},
{
"oligo_id": "illumina_p5_adapter",
"name": "Illumina P5 adapter",
"aliases": [],
"role": "P5 flow-cell binding sequence (5' end of final library)",
"kind": "single",
"sequence": "AATGATACGGCGACCACCGAGATCTACAC",
"direction": "5_to_3",
"components": [],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Introduced via RP1; canonical Illumina P5."
},
{
"oligo_id": "illumina_p7_adapter",
"name": "Illumina P7 adapter",
"aliases": [],
"role": "P7 flow-cell binding sequence (3' end of final library)",
"kind": "single",
"sequence": "CAAGCAGAAGACGGCATACGAGAT",
"direction": "5_to_3",
"components": [],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Introduced via RPIX; canonical Illumina P7. Appears in the final library as its reverse complement ATCTCGTATGCCGTCTTCTGCTTG."
}
],
"final_library": {
"source_label": "CEL-Seq final Illumina (TruSeq Small RNA) library",
"annotated_library_sequence": "AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC[CELL_BARCODE:8]TTTTTTTTTTTTTTTTTTTTTTTT[CDNA]TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC[SAMPLE_INDEX:6]ATCTCGTATGCCGTCTTCTGCTTG",
"library_sequence": "AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTXXXXXXXXXXTGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG",
"strands": [
{
"direction": "5_to_3",
"source_html": "AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG",
"source_sequence": "AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG"
}
],
"annotation_lines": [
"AATGATACGGCGACCACCGAGATCTACAC = P5",
"GTTCAGAGTTCTACAGTCCGACGATC = Illumina 5' RNA adapter (RA5) / read 1 region",
"[CELL_BARCODE:8] = Cell barcode (CEL-Seq RT-primer barcode)",
"TTTTTTTTTTTTTTTTTTTTTTTT = poly(dT) (mRNA polyA-derived)",
"[CDNA] = cDNA insert (3'-most fragment of the transcript)",
"TGGAATTCTCGGGTGCCAAGG = Illumina 3' RNA adapter (RA3)",
"AACTCCAGTCAC = TruSeq Small RNA read 2 / RP linker",
"[SAMPLE_INDEX:6] = i7 sample index (RPIX)",
"ATCTCGTATGCCGTCTTCTGCTTG = reverse complement of P7"
],
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "CEL-Seq final Illumina (TruSeq Small RNA) library",
"method": "claude_llm_extraction"
}
]
},
"read_structure": {
"reads": [
{
"read": "R1",
"primer": "Illumina small-RNA Read 1 sequencing primer (RA5)",
"template": "bottom",
"cycles": 15,
"segments": [
{
"name": "Cell barcode",
"type": "barcode",
"order": 0,
"scored": true,
"provenance": null,
"whitelist_ref": null,
"constant_ref": null,
"notes": null,
"length": 8
},
{
"name": "poly(dT)",
"type": "polyA",
"order": 1,
"scored": false,
"provenance": null,
"whitelist_ref": null,
"constant_ref": null,
"notes": null,
"length": 7
}
]
},
{
"read": "I1",
"primer": "Illumina index sequencing primer",
"template": "top",
"cycles": 6,
"segments": [
{
"name": "i7 sample index",
"type": "index",
"order": 0,
"scored": false,
"provenance": null,
"whitelist_ref": null,
"constant_ref": null,
"notes": null,
"length": 6
}
]
},
{
"read": "R2",
"primer": "Illumina small-RNA Read 2 sequencing primer (RA3/RP)",
"template": "top",
"cycles": 50,
"segments": [
{
"name": "cDNA insert",
"type": "insert",
"order": 0,
"scored": true,
"provenance": null,
"whitelist_ref": null,
"constant_ref": null,
"notes": null
}
]
}
]
},
"library_generation": [
{
"step": 1,
"title": "Single-cell lysis, primer annealing & first-strand synthesis",
"summary": "The barcoded CEL-Seq primer captures the cell's polyA mRNA and ArrayScript reverse transcriptase copies it into first-strand cDNA.",
"note": "Each frozen single cell receives one uniquely barcoded CEL-Seq RT primer (+ERCC spike-in), is heated to open the cell and anneal the anchored poly(dT), then reverse transcribed at 42C (Ambion MessageAmp II ArrayScript).",
"product": " T7 promoter RA5 (5' adapter) BC poly(dT)\n5'- CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATC[CELL_BARCODE:8]TTTTTTTTTTTTTTTTTTTTTTTTV[CDNA]------->\n 3'- ...AAAAAAAAAA[mRNA] -5'"
},
{
"step": 2,
"title": "Second-strand synthesis",
"summary": "RNaseH + DNA Pol I convert the cDNA:mRNA hybrid into double-stranded cDNA carrying a functional T7 promoter.",
"note": "Standard second-strand reaction at 16C yields dsDNA; the T7 promoter is now double-stranded and transcription-competent.",
"product": "5'- CGATTGAGGCCGGTAATACGACTCACTATAGGGGTTCAGAGTTCTACAGTCCGACGATC[CELL_BARCODE:8]TTTTTTTTTTTTTTTTTTTTTTTTV[CDNA] -3'\n3'- GCTAACTCCGGCCATTATGCTGAGTGATATCCCCAAGTCTCAAGATGTCAGGCTGCTAG[CELL_BARCODE:8]AAAAAAAAAAAAAAAAAAAAAAAAB[CDNA] -5'"
},
{
"step": 3,
"title": "Pooling + T7 in vitro transcription (linear amplification)",
"summary": "Barcoded double-stranded cDNAs from many cells are pooled and linearly amplified in one round of T7 IVT into antisense amplified RNA (aRNA).",
"note": "Pooling reaches the ~400 pg IVT input threshold; a single 13 hr IVT reaction makes many antisense RNA copies per molecule, each retaining the barcode and 5' adapter.",
"product": "aRNA (antisense, 5'->3'; hundreds of copies):\n5'- GGGUUCAGAGUUCUACAGUCCGACGAUC[CELL_BARCODE:8]UUUUUUUUUUUUUUUUUUUUUUUU[antisense-cDNA] -3'"
},
{
"step": 4,
"title": "RNA fragmentation",
"summary": "aRNA is chemically fragmented to a sequencing-appropriate size, peaking around 500 nt.",
"note": "Fragmentation (Tris-acetate/KOAc/MgOAc, 94C, 3 min) breaks aRNA; only the 5'-most fragment still carries the barcode + RA5 adapter.",
"product": "5'- GGGUUCAGAGUUCUACAGUCCGACGAUC[CELL_BARCODE:8]UUUUUUUUUUUU[frag] -3' (barcode-bearing fragment)\n5'- [internal aRNA fragments, no adapter] -3' (not retained in final library)"
},
{
"step": 5,
"title": "End repair (Antarctic phosphatase + PNK)",
"summary": "Fragment ends are dephosphorylated then re-phosphorylated to give 5'-monophosphate / 3'-OH ends compatible with adapter ligation.",
"note": "Antarctic phosphatase removes 3' phosphates/cyclic phosphates; T4 PNK adds a 5' phosphate, priming the RNA for T4 RNA ligase 2 (truncated).",
"product": "5'-P- GGGUUCAGAGUUCUACAGUCCGACGAUC[CELL_BARCODE:8]UUUUUUUUUUUU[frag] -OH-3'"
},
{
"step": 6,
"title": "3' adapter (RA3) ligation",
"summary": "The Illumina 3' small-RNA adapter (RA3) is ligated onto the 3' end of the aRNA fragments.",
"note": "T4 RNA Ligase 2 (truncated) attaches RA3 (diluted 1:5) to 3'-OH ends; only fragments with both the 5' (RA5, from the primer) and 3' (RA3) adapters become amplifiable.",
"product": "5'- GGGUUCAGAGUUCUACAGUCCGACGAUC[CELL_BARCODE:8]UUUUUUUUUUUU[frag]TGGAATTCTCGGGTGCCAAGG -3'"
},
{
"step": 7,
"title": "Reverse transcription (RTP primer)",
"summary": "The RNA RT primer (RTP) anneals to the ligated RA3 adapter and SuperScript II copies the adapter-flanked RNA into cDNA.",
"note": "RTP is complementary to RA3; RT at 50C generates a cDNA copy that is now the PCR template.",
"product": "RNA: 5'- ...GTTCAGAGTTCTACAGTCCGACGATC[CELL_BARCODE:8](T)24[frag]TGGAATTCTCGGGTGCCAAGG -3'\nRTP: 3'- ACCTTAAGAGCCCACGGTTCCG -5'\ncDNA: 3'- ...CAAGTCTCAAGATGTCAGGCTGCTAG[CELL_BARCODE:8](A)24[frag]ACCTTAAGAGCCCACGGTTCCG -5'"
},
{
"step": 8,
"title": "Library PCR (RP1 + indexed RPIX) & bead cleanup",
"summary": "12 cycles of PCR with RP1 (P5) and an indexed RPIX (i7 + P7) complete both flow-cell adapters and add the library index, producing the final dsDNA library.",
"note": "RP1 adds P5/completes RA5; RPIX adds the 6-nt i7 index and P7. Two AMPure XP cleanups yield a ~300-400 bp library.",
"product": "5'- AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC[CELL_BARCODE:8]TTTTTTTTTTTTTTTTTTTTTTTT[CDNA]TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC[SAMPLE_INDEX:6]ATCTCGTATGCCGTCTTCTGCTTG -3'"
}
],
"library_sequencing": [
{
"read": "Read 1 (cell barcode + poly(T))",
"primer": "Illumina small-RNA Read 1 sequencing primer (sequence = RA5; anneals to the bottom strand across the 5' RNA adapter)",
"template": "bottom",
"cycles": 15,
"note": "Reads the 8-bp cell barcode immediately followed by the anchored poly(T) stretch (>=15 bases were read for read 1); >95% of reads carry the barcode precisely at the start of read 1, invariably followed by poly(T).",
"diagram": "Read 1 primer (= RA5) anneals to bottom strand; reads cell barcode then poly(T):\n5'-...GTTCAGAGTTCTACAGTCCGACGATC-----------------> (read 1, 5'->3')\n5'-...GTTCAGAGTTCTACAGTCCGACGATCNNNNNNNNTTTTTTT...[cDNA]-3' (top)\n3'-...CAAGTCTCAAGATGTCAGGCTGCTAGNNNNNNNNAAAAAAA...[cDNA]-5' (bottom = template)\n ^cell BC(8)^ ^poly(T)^"
},
{
"read": "Index 1 (i7 sample index)",
"primer": "Illumina i7 index sequencing primer (anneals across the RP/RA3 linker on the top strand, 3' end abutting the index)",
"template": "bottom",
"cycles": 6,
"note": "Reads the 6-nt library-level index carried on the indexed RNA PCR primer (RPIX); used to demultiplex multiple IVT pools sequenced on the same lane. Read is only taken when multiplexing IVT pools.",
"diagram": "i7 index primer anneals over RP linker; reads the 6-nt sample index:\n5'-...TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC------> (index read, 5'->3')\n5'-...TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG-3' (top)\n3'-...ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC-5' (bottom = template)\n ^i7 index(6)^"
},
{
"read": "Read 2 (cDNA / mRNA transcript)",
"primer": "Illumina small-RNA Read 2 sequencing primer (sequence complementary to RA3; anneals to the top strand across the 3' RNA adapter)",
"template": "top",
"cycles": 50,
"note": "Reads 50 bases into the 3'-most cDNA fragment, identifying the transcript strand-specifically (>97% of exonic reads sense-oriented; reads map to transcript 3' ends).",
"diagram": "Read 2 primer anneals over RA3 on top strand; reads into 3'-most cDNA fragment (antisense):\n 3'-ACCTTAAGAGCCCACGGTTCC-5' (Read 2 primer)\n <------(read 2)\n5'-...TTTTTTT[.....cDNA insert.....]TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC...-3' (top = template)\n3'-...AAAAAAA[.....cDNA insert.....]ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTG...-5' (bottom)"
}
],
"whitelists": {},
"build": {
"builder_version": "llm-generic-1.0",
"deterministic": false,
"source_html_sha256": null,
"extraction_method": "claude_llm_generic",
"model": "claude-opus-4-8"
},
"title": "CEL-Seq",
"description": "CEL-Seq (Cell Expression by Linear amplification and Sequencing) is a multiplexed single-cell RNA-seq method that barcodes each cell's mRNA during reverse transcription, pools the samples, and linearly amplifies them with a single round of T7 in vitro transcription (IVT) instead of PCR. Each cell's RT primer carries an anchored poly(dT), an 8-bp cell barcode, the Illumina 5' small-RNA adapter, and a T7 promoter. After IVT the amplified antisense RNA is fragmented and built into an Illumina directional (TruSeq Small RNA) library. Paired-end sequencing reads the cell barcode followed by a poly(T) stretch in read 1 and the 3'-most mRNA fragment in read 2, yielding strand-specific, 3'-end digital gene-expression counts.",
"reference": {
"kind": "protocol_doc",
"label": "cel seq",
"path": null,
"url": null,
"doi": null
},
"publication": {
"year": 2012,
"original_publication": {
"title": "CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification",
"journal": "Cell Reports",
"doi": "10.1016/j.celrep.2012.08.003",
"url": "http://dx.doi.org/10.1016/j.celrep.2012.08.003"
},
"authors": [
{
"name": "Tamar Hashimshony",
"affiliation": "Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel"
},
{
"name": "Florian Wagner",
"affiliation": "Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel"
},
{
"name": "Noa Sher",
"affiliation": "Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel"
},
{
"name": "Itai Yanai",
"corresponding": true,
"email": "yanai@technion.ac.il",
"affiliation": "Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel"
}
],
"throughput": {
"summary": "Amplification of up to ~50 cells per person per day; ~10 IVT pools (~500 cells) converge into a single library preparation. The 8-bp barcode design makes the number of uniquely barcoded samples per IVT essentially unlimited.",
"cells": "~500 single cells per library prep",
"rna": "IVT requires ~400 pg total RNA input; RT works down to ~5 pg (single mammalian cell). 10 pg C. elegans RNA ~ 280,000 mRNA molecules.",
"dna": "Final library ~1 ng/ul, size peak ~300-400 bp"
},
"statistical_model": "Absolute molecule counts obtained by least-squares linear regression on ERCC spike-in expression levels; per-gene expression reported as transcripts per million (tpm), with differential expression tested by FDR-corrected t tests.",
"other": [
{
"label": "NCBI SRA accession",
"value": "SRP014672"
},
{
"label": "Amplification",
"value": "Single round of linear T7 in vitro transcription (IVT), not PCR"
},
{
"label": "Strand specificity",
"value": ">98% of exonic reads from the sense strand"
},
{
"label": "Barcoding efficiency",
"value": ">96% of reads contain a barcode (95.5% at start of read 1)"
},
{
"label": "rRNA contamination",
"value": "<2% of reads (polyA-specific)"
},
{
"label": "Kits",
"value": "Ambion MessageAmp II aRNA (AM1751) + Illumina TruSeq Small RNA sample prep (RS-200-0012)"
},
{
"label": "Sequencer",
"value": "Illumina HiSeq2000, paired-end"
}
]
},
"modality": "RNA",
"method_type": "manual/tube",
"data_processing": {
"summary": "A custom multistep pipeline run in the Galaxy framework: paired-end reads are trimmed/filtered, demultiplexed by the first 8 bases of read 1 (the cell barcode), read 2 is mapped to the reference genome, reads are counted per gene and normalized to transcripts per million (tpm). Because CEL-Seq retains a single 3'-most fragment per transcript, counts require no gene-length normalization; absolute mRNA copy numbers are then obtained by least-squares linear regression on the ERCC spike-in values.",
"stages": [
{
"id": "preprocess",
"label": "Read preprocessing"
},
{
"id": "demultiplex",
"label": "Demultiplexing"
},
{
"id": "align",
"label": "Alignment"
},
{
"id": "quantify",
"label": "Quantification"
},
{
"id": "normalize",
"label": "Normalization & absolute counts"
}
],
"nodes": [
{
"id": "trim",
"label": "Trim reads",
"tool": "Galaxy",
"stage": "preprocess",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "filter",
"label": "Filter reads",
"tool": "Galaxy",
"stage": "preprocess",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "demux",
"label": "Demultiplex by cell barcode (8 bp)",
"tool": "custom scripts",
"stage": "demultiplex",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "map",
"label": "Map read 2 to genome",
"tool": "",
"stage": "align",
"scope": "per_cell",
"terminal": false,
"viz_only": false
},
{
"id": "count",
"label": "Count reads per gene",
"tool": "htseq-count",
"stage": "quantify",
"scope": "per_cell",
"terminal": false,
"viz_only": false
},
{
"id": "tpm",
"label": "Normalize to tpm",
"tool": "custom scripts",
"stage": "normalize",
"scope": "per_cell",
"terminal": false,
"viz_only": false
},
{
"id": "absolute",
"label": "Convert tpm to absolute copies",
"tool": "custom scripts",
"stage": "normalize",
"scope": "per_cell",
"terminal": true,
"viz_only": false
}
],
"edges": [
{
"from": "trim",
"to": "filter",
"kind": "sequential"
},
{
"from": "filter",
"to": "demux",
"kind": "sequential"
},
{
"from": "demux",
"to": "map",
"kind": "sequential"
},
{
"from": "map",
"to": "count",
"kind": "sequential"
},
{
"from": "count",
"to": "tpm",
"kind": "sequential"
},
{
"from": "tpm",
"to": "absolute",
"kind": "sequential"
}
],
"statistical_model": "Least-squares linear regression of tpm-normalized counts against known ERCC spike-in concentrations to recover absolute mRNA copy numbers; no gene-length normalization is applied because CEL-Seq retains a single 3'-most fragment per transcript."
}
}
|