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Sample GSM4987660

Query DataSets for GSM4987660

Status

Public on Dec 23, 2023

Title

Control_d12_rep2

Sample type

SRA

Source name

Control 3T3-L1 adipocyte, day 12 (d12) after differentiation

Organism

Mus musculus

Characteristics

cell line: 3T3-L1 adipocyte
genotype: Control
time: day 12 (d12) after differentiation

Treatment protocol

Day 0 (d0) is set when the media was replaced to the adipocyte differentiation media. The cells were harvested at d0, d6 and d12.

Growth protocol

The control or Pax7 stably expressed 3T3-L1 adipocytes were cultured with 10% FBS DMEM medium on 35mm dish until the cells reached to confluent. They were kept for two days with the confluency. The media was changed to 3T3-L1 adipocyte differentiation medium for three days and to adipocyte maintenance medium for nine days.

Extracted molecule

total RNA

Extraction protocol

Total RNA was obtained from the 3T3-L1 cells using NucleoSpin RNA XS (Takara) .
For CEL-Seq2, sequencing was performed using HiSeq PE Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2 (50 Cycles) on Illumina HiSeq 1500 sequencers with the following cycles: 15 cycles for read 1 and 45 cycles for read 2. for ChIP-seq. For ChIP-Seq2, the libraries were sequenced on Illumina HiSeq 1500 sequencers using the Hiseq SR Cluster Kit v4-cBot and Hiseq SBS Kit v4 (50cycles), and on Illumina NovaSeq 6000 sequencers using NovaSeq 6000 SP Reagent Kit (100 Cycles).
The cells were harvested at d0, d6 and d12, then extracted RNA was utilized for RNAseq by CEL-Seq2. The 3T3-L1 cells at d0 were used for the ChIP-seq analysis.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Description

CEL0428raw_Izumi_Pax73T3_countmatrix_genename.csv
CEL0428raw_Izumi_Pax73T3_normalizedcount_genename.csv

Data processing

For RNA-seq, extraction of sample barcodes and unique-molecular-identifiers (UMIs) was performed using UMI-tools (version 1.0.1) with the following commands: umi_tools extract -I r1.fastq --read2-in=r2.fastq --bc-pattern=NNNNNNCCCCCC --read2-stdout.
The reads were trimmed using Cutadapt (M. Martin, 2011) (version 2.6) wapper, Trim Galore! (version 0.6.6), with the option: -a GATCGTCGGACT. The reads were mapped by the aligning software HISAT2 (D. Kim et al., 2015, M. Pertea et al., 2016, D. Kim et al., 2019) (version 2.1.0) to the reference genome (GRCm38, pre-built HISAT2 index, genome_snp_tran).
Read counts per gene were obtained using featureCounts (Liao Y et al., 2014) (version 2.0.1). Note that UMIs were not used and mitochondrial chromosome genes are excluded in the analysis.
For ChIL-seq analysis, raw sequencing data were trimmed using Cutadapt (version 2.6) wapper, Trim Galore! (version 0.6.6).
Bowtie2 (B. Langmead et al., 2012) (version 2.3.5.1) was used to map the raw sequence reads (fastq) to the mouse mm10 reference genome. Note that the uniquely mapped reads were retained.
The software BEDtools (A. R. Quinlan et al., 2010) (version 2.29.2) with “multicov” option was used to count reads of ChIP-seq within 2.5kb±TSS regions of the first exons.
For RNA-seq and ChIP-seq,Downstream analysis was conducted by DESeq2 software (M. I. Love et al., 2014) (1.28.1).
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: ChIP-seq signal tracks (bigWig) were created using the software deepTools (Ramírez et al., 2016) (version 3.3.0) with the options: bamCoverage --binSize 100 --smoothLength 1000 --normalizeUsing CPM.
Supplementary_files_format_and_content: *.csv file include comma-delimited RNA-seq read count matrix and normalized count matrix.

Submission date

Dec 23, 2020

Last update date

Dec 23, 2023

Contact name

Yasuyuki Ohkawa

E-mail(s)

yohkawa@bioreg.kyushu-u.ac.jp

Organization name

Medical Institute of Bioregulation

Lab

Division of Transcriptomics