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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 03, 2024 |
| Title |
KO-rep1 [scRNA-Seq] |
| Sample type |
SRA |
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| Source name |
mouse HSC
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| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow
genotype: Mat KO
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| Treatment protocol |
FACS-sorted HSC were kept on ice until lysed and reverse transcribed.
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| Extracted molecule |
total RNA |
| Extraction protocol |
modified STRT-seq
A modified STRT-seq protocol was applied for single-cell RNA-seq. Briefly, sorted single cells in good condition were picked into lysis by mouth pipetting, and the scRNA-seq libraries were constructed based on STRT-seq with some modifications. cDNAs were synthesized using sample-specific 25-nt oligo-dT primer containing 8-nt barcode (TCAGACGTGTGCTCTTCCGATCT-XXXXXXXX-DDDDDDDD-T25, X representing sample-specific barcode while D standing for unique molecular identifiers (UMI). After reverse transcription and second-strand cDNA synthesis, the cDNAs were amplified by 16 cycles of PCR using ISPCR primer and 3’ Anchor primer. Samples were pooled and purified using Agencourt AMPure XP beads . 4 cycles of PCR were performed to introduce index sequence and subsequently, 400 ng cDNAs were fragmented to around 300 bp by Covaris S2. After being incubated with Dynabeads MyOneTM Streptavidin C1 beads (Thermo Fisher, 65002) for 1 hour at room temperature, cDNA Libraries were generated using KAPA Hyper Prep Kit (Kapa Biosystems, kk8505). After adaptor ligation, the libraries were amplified by 8 cycles of PCR using QP2 primer and short universal primer. The libraries were sequenced on Illumina Hiseq 4000 platform in 150 bp pair-ended manner
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
KO1
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| Data processing |
Illumina Casava1.8 software used for basecalling.
For modified STRT-seq data, raw reads were first split for each cell by specific barcode sequence attached in Read 2.
The TSO sequence and polyA tail sequence were trimmed for the corresponding Read 1 after UMI information was aligned to it. Reads with adapter contaminants or low-quality bases (N > 10%) were discarded.
Subsequently, we aligned the stripped Read 1 sequences to mm10 mouse transcriptome (UCSC) using tophat2 (version 2.0.12). Uniquely mapped reads were counted by HTSeq package and grouped by the cell-specific barcodes.
Duplicated transcripts were removed based on the UMI information for each gene. Finally, for each individual cell, the copy number of transcripts of a given gene was the number of the distinct UMIs of that gene.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files with raw gene umi.
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| Submission date |
Feb 10, 2021 |
| Last update date |
Jun 03, 2024 |
| Contact name |
Jiang Penglei |
| E-mail(s) |
jpl3982233@zju.edu.cn
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| Organization name |
Zhejiang University
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| Department |
School of basic medicine science
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| Street address |
Yuhangtang Road 866
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| City |
Hangzhou |
| ZIP/Postal code |
310058 |
| Country |
China |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE166516 |
Small Nucleolar RNAs in the Dlk1-Gtl2 Imprinting Locus Maintains LT-HSC Self-renewal by Regulation of rRNA Modification and Translation [scRNA-Seq] |
| GSE166518 |
Small Nucleolar RNAs in the Dlk1-Gtl2 Imprinting Locus Maintains LT-HSC Self-renewal by Regulation of rRNA Modification and Translation |
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| Relations |
| BioSample |
SAMN17850048 |
| SRA |
SRX10064677 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5073639_KO1.refgene.umi.txt.gz |
345.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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