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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jun 26, 2023 |
Title |
P12_40d_Nr2f2-KO_88, scRNA-seq |
Sample type |
SRA |
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Source name |
liver
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Organism |
Mus musculus |
Characteristics |
tissue: liver cell type: hepatocyte genotype: Rosa-Cas9 age: P12+40d ploidy: / treatment: Nr2f2-KO zonation: /
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Extracted molecule |
total RNA |
Extraction protocol |
For scRNA-seq, single cells were picked up into cell lysis buffer, and mRNA was reverse transcribed and amplified. For snRNA-seq, nucleus extraction was performed as previously reported(Krishnaswami et al., 2016). In brief, hepatocytes were collected through FACS sorting. Cell membranes were broken down with a homogenizer in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris buffer (pH 8.0), 1 µM DTT, protease inhibitor, RNase inhibitor and 0.1% Triton X-100). After centrifugation (5 min, 500 g, 4° C), the nucleus was washed with PBS containing RNase inhibitor and stained with 0.5 ug/ml DAPI. The single nucleus was sorted into cell lysis. SnRNA-seq was performed using well-based mSTRT-seq just as scRNA-seq, but the cycles of cDNA amplification was 4 + 16. For bulk RNA-seq, total RNA was extracted using the RNAprep Pure Micro Kit. Poly(A) RNA was purified with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490). For well-based mSTRT-seq(Li et al., 2017), barcoded DNA products were pooled and libraries were constructed with a Kapa Hyper Prep Kit (Kapa Biosystems, KK8505). For Smart-seq3(Hagemann-Jensen et al., 2022), the library of each cell was constructed separately with TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501). For bulk RNA-seq, libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads (NEB, #E7765) according to the manufacturer’s instructions.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
MGISEQ-2000RS |
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Description |
MGI-seq 2000 Smart-seq3 readcount.Smartseq3.csv
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Data processing |
For mSTRT-seq, libraries were sequenced as 150-bp paired-end reads on an Illumina HiSeq 4000 platform. Raw files (fastq format) were separated based on cell-specific barcode sequences. PolyA sequences were trimmed from R1 reads (3’ end of cDNA). Preprocessed R1 reads from each cell were then aligned to the Mus musculus reference genome GRCm38/mm10 with TopHat V2.1.0. Bam files were annotated to genes with featureCounts (v1.5.3). We used samtools (v1.3.1) to sort and index the output bam files. UMIs of each gene were counted with umi-tools (v0.5.0) with the “unique” method. For Smart-seq 3, libraries were sequenced as 100-bp paired-end reads on an MGI-seq 2000 platform. We trimmed the index, primer and adaptor sequences from the paired-end reads using Trimmomatic v0.39. The trimmed reads were aligned to the M. musculus reference genome GRCm38/mm10 with HISAT v2.1.0. Bam files were annotated to genes with featureCounts (v2.0.0). Either 5’ fragments or internal fragments were used during alignment. For snRNA-seq, libraries were sequenced as 150-bp paired-end reads on an Illumina HiSeq 4000 platform. The alignment processes of snRNA-seq were similar to those of mSTRT-seq. The specific parameter of featureCounts (v1.5.3) during snRNA-seq alignment was “-t transcript” to ensure that the intron of mRNA could be detected. For RNA-seq, libraries were sequenced as 150-bp paired-end reads on an Illumina HiSeq 2500 platform. the sequencing reads were aligned to the M. musculus genome (mm10) using TopHat (v2.1.0; https://ccb.jhu.edu/software/tophat/index.shtml) with the parameters ‘-o out_dir -G gtf –transcriptome-index trans_index bowtie2_index input_fastq1 input_fastq2’. Reads aligned to genes were counted using HTSeq (v0.6.0; https://htseq.readthedocs.io/) with the parameters ‘htseq-count -f bam -r pos -s reverse -a 30 input_bam gtf’. Assembly: mm10 Supplementary files format and content: Comma-Separated Values files (.csv)
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Submission date |
Jul 25, 2022 |
Last update date |
Jun 26, 2023 |
Contact name |
Cheng-ran Xu |
Organization name |
Peking University
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Department |
School of Basic Medical Sciences
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Street address |
NO.5 YIHEYUAN ROAD HAIDIAN DISTRICT, BEIJING, P.R.CHINA
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City |
Beijing |
State/province |
- |
ZIP/Postal code |
100871 |
Country |
China |
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Platform ID |
GPL30215 |
Series (1) |
GSE209749 |
scRNA-seq, snRNA-seq and RNA-seq of mouse hepatocytes during liver maturation |
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Relations |
BioSample |
SAMN30007935 |
SRA |
SRX16709589 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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