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| Status |
Public on Jan 26, 2022 |
| Title |
PSCSR-seq analysis for whole cells and nuclei |
| Sample type |
SRA |
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| Source name |
cultured cell line
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell line: HEK293T and 3T3
passages: 15-20
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| Growth protocol |
A549 cells were cultured in DMEM/F-12 (11320082, Gibco), Hela, 3T3, and HEK293T cells were cultured in DMEM-basic (C11965500BT, Gibco) and K562 cells were cultured in RPMI Medium 1640 basic(1X) (C22400500BT,Gibco). All the cultured media were supplemented with 10% (v/v) foetal bovine serum (26140079, Gibco) and 1% penicillin-streptomycin (15140122, Gibco). Cells were cultured at 37°C in a 5% CO2 humidified incubator.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cultured cancer cells, PBMCs, and tumor tissue were harvested and dissociated. Cells were dispensed into a ICELL8 nanowell chip for PSCSR-seq analysis.
single cell small RNA analysis used PSCSR-seq protocol, briefly, small RNAs were ligated with 3' adapters using T4 RNA Ligase 2, truncated KQ (NEB), the remained 3' adapter were removed by 5' Deadenylase (NEB) and Lambda Exonuclease (Thermo Fisher). 5'adapters were ligated to the small RNA using T4 RNA Ligase 1(NEB). The adapters carrying 8 random nucleotides (UMI) for the small RNA counting. The ligated small RNAs were reverse-transcribed using Superscript III (Thermo Fisher), and two rounds of PCRs were performed to introduce the barcodes for labeling cells and multiplexing samples. PSCSR library was size selected with Pippin Prep, and the size distribution was assessed using an Agilent 2100 BioAnalyzer; Single cell RNA-seq followed Chromium Single Cell 3' Reagent Kits User Guide (v2 Chemistry) from the 10X Genomics company.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
PSCSR-seq results for 293T whole cells, 293T nuclei, 3T3 whole cells and 3T3 nuclei
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| Data processing |
The PSCSR-seq analysis procedures are adapted from the method in the publication(S. Mi and T. Cai et al., Cell 133, 116, 2008). Briefly, the small RNA sequences, cell barcodes and molecules UMI are extracted from the raw reads using the customized scripts. The small RNA sequence with the length of 16nt~39nt were kept and mapped to the reference genome with no mismatch (bowtie v1.2.2). The small RNAs are annotated to according to the databases including miRbase (release v22.1), EBI RNAcenter database (release v11), EBML CDS database (ENSEMBL release v96). For certain small RNA gene, different UMIs (at least 2-base difference) were counted as the measurement of small RNA expression. The computational pipeline can be found at github (https://github.com/biocaitao/PSCSR-seq).
The single cells in PSCSR-seq were selected using TaKaRa ICell8 system protocol (“StandardCellSettings-V5” or “PBMCv4”). The cell barcodes of selected cells were exported. The valid cells were filtered from the background according to the exported barcodes (allowing 2 mismatches). (refer to https://github.com/biocaitao/PSCSR-seq ).
Genome_build: hg38 and mm10
Supplementary_files_format_and_content: PSCSR-seq processed files contain: plain text files for cell barcodes and estimated expression of miRNAs and all small RNAs;
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| Submission date |
Jan 26, 2022 |
| Last update date |
Jan 27, 2022 |
| Contact name |
Tao Cai |
| E-mail(s) |
caitao@nibs.ac.cn
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| Organization name |
National Institute Of Biological Sciences, Beijing (NIBS)
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| Lab |
Sequencing Facility
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| Street address |
No. 7 Science Park Road, Zhongguancun Life Science Park
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| City |
Beijing |
| ZIP/Postal code |
102206 |
| Country |
China |
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| Platform ID |
GPL25526 |
| Series (1) |
| GSE134004 |
Small RNA transcriptome analysis by parallel single cell small RNA sequencing (PSCSR-seq) |
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| Relations |
| BioSample |
SAMN25282623 |
| SRA |
SRX13932323 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5838225_293T_miRNA.txt.gz |
974.2 Kb |
(ftp)(http) |
TXT |
| GSM5838225_293T_sRNA.txt.gz |
6.7 Mb |
(ftp)(http) |
TXT |
| GSM5838225_3T3_miRNA.txt.gz |
980.8 Kb |
(ftp)(http) |
TXT |
| GSM5838225_3T3_sRNA.txt.gz |
6.9 Mb |
(ftp)(http) |
TXT |
| GSM5838225_nuclei_barcodes.txt.gz |
4.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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