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Sample GSM6393608

Query DataSets for GSM6393608

Status

Public on Jun 26, 2023

Title

P12_40d_Nr2f2-KO_93, scRNA-seq

Sample type

SRA

Source name

liver

Organism

Mus musculus

Characteristics

tissue: liver
cell type: hepatocyte
genotype: Rosa-Cas9
age: P12+40d
ploidy: /
treatment: Nr2f2-KO
zonation: /

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MGISEQ-2000RS

Description

MGI-seq 2000
Smart-seq3
readcount.Smartseq3.csv

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)

Submission date

Jul 25, 2022

Last update date

Jun 26, 2023

Contact name

Cheng-ran Xu

Organization name

Peking University

Department

School of Basic Medical Sciences

Street address

NO.5 YIHEYUAN ROAD HAIDIAN DISTRICT, BEIJING, P.R.CHINA