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| Status |
Public on Aug 17, 2017 |
| Title |
frozen fixed HEK293T and NIH/3T3 cell mixture (16 x 80 sci-RNA-seq) |
| Sample type |
SRA |
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| Source name |
cell line
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell line: HEK293T and NIH/3T3
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| Treatment protocol |
For c.elegans, a synchronized L2 population was obtained by two cycles of bleaching gravid adults to isolate fertilized eggs allowing the eggs to hatch in the absence of food to generate a population of starved L1 animals.
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| Growth protocol |
All cultured cell lines were cultured at 37°C with 5% CO2, and were maintained in high glucose DMEM (Gibco cat. no. 11965) supplemented with 10% FBS and 1X Pen/Strep (Gibco cat. no. 15140122; 100U/ml penicillin, 100µg/ml streptomycin).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
All cell lines were trypsinized, spun down at 300xg for 5 min (4°C). and washed once in 1X PBS. L2 stage worms were disscociated with SDS-DTT solution and pronase to generate single cell suspension. Detailed protocol was described in "J. Cao et al., Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing. bioRxiv, 104844 (2017)."
library construction protocol was described in "J. Cao et al., Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing. bioRxiv, 104844 (2017)."
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
sciRNAseq with HEK293T and NIH/3T3 cells (cells are methanol fixed and frozen in -80 degree for 4 days)
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| Data processing |
Base calls were converted to fastq format and demultiplexed using Illumina’s bcl2fastq/ 2.16.0.10 tolerating one mismatched base in barcodes (edit distance (ED) < 2)
RT barcode (10bp) and UMI (8bp) sequence in Read1 is attached to the read name of Read2. Reads not matching to the expected RT barcodes were discarded. Read2 were then adaptor clipped using trim_galore/0.4.1 with default settings.
Trimmed reads were mapped to the human reference genome (hg19), mouse reference genome (mm10), C.elegans reference genome (PRJNA13758) or a chimeric reference genome of hg19, mm10 and PRJNA13758, using STAR/v 2.5.2b (38) with default settings and gene annotations (GENCODE V19 for human; GENCODE VM11 for mouse, WormBase PRJNA13758.WS253.canonical_gene set for C.elegans).
Uniquely mapping reads were extracted, and duplicates were removed using the unique molecular identifier (UMI) sequence (ED < 2, including insertions and deletions), reverse transcription (RT) index, and read 2 end-coordinate.
Mapped reads were split into constituent cellular indices by further demultiplexing reads using the RT index (ED < 2, including insertions and deletions).
To generate digital expression matrices, we calculated the number of strand-specific UMIs mapping to the exonic and intronic regions of each gene, for each cell; generally, fewer than 3% of total UMIs strand-specifically mapped to multiple genes. For multi-mapped reads, reads were assigned to the closest gene, except in cases where another intersected gene fell within 100 bp to the end of the closest gene, in which case the read was discarded.
Genome_build: human reference genome (hg19), mouse reference genome (mm10), C.elegans reference genome (PRJNA13758)
Supplementary_files_format_and_content: Processed data file is a sparsematrix in R: each row corresponds to genes, and each column corresponds to single cell, number in the matrix is the UMI count data.
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| Submission date |
May 04, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Junyue Cao |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Department |
Department of Genome Sciences
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| Lab |
Shendure lab
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| Street address |
Foege Building S-210, 3720 15th Ave NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195-5065 |
| Country |
USA |
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| Platform ID |
GPL19415 |
| Series (1) |
| GSE98561 |
Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing |
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| Relations |
| BioSample |
SAMN06891877 |
| SRA |
SRX2784964 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2599704_Gene.count.matrix.human.mouse.4.Rdata.gz |
3.5 Mb |
(ftp)(http) |
RDATA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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