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Sample GSM4417240

Query DataSets for GSM4417240

Status

Public on Jun 23, 2020

Title

SCC1_diff_auxin_RNA_rep2

Sample type

SRA

Source name

SCC1_diff_auxin_noselection_RNA

Organism

Mus musculus

Characteristics

auxin treatment: yes
genotype: SCC1 KO

Treatment protocol

SCC1-AID-GFP mESCs were cultured in medium without LIF for 3 days and treated without/with Auxin (500 μM) for 1day. The differentiated cells were dissociated into single cells. Sorting was then conducted using FACS Jazz to remove all the dead cells. Cells depleted of GFP fluorescence signals in auxin treatment group were collected as SCC1 KO (SCC1 -) groups, while the differentiated cells without the auxin treatment were collected as SCC1 WT (SCC1 +) groups.

Extracted molecule

polyA RNA

Extraction protocol

The RNA extraction follows smart-seq2 protocol
The RNA-seq libraries were generated using the Smart-seq2 protocol as described previously with minor modification (Picelli et al., 2014). Cells were lysed in hypotonic lysis buffer (Amresco, M334), and the polyadenylated mRNAs were captured by the PolyT primers. After ~3–10 min lysis at 72 °C, the Smart-seq2 reverse transcription reactions were performed. After pre-amplification and AMPure XP beads purification, cDNAs were sheared by Covaris and were subject to Illumina TruSeq library preparation. All libraries were sequenced on Illumina HiSeq 2500 according to the manufacturer’s instruction.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

SCC1_diff_facs_select_noselect_fpkm.txt.gz

Data processing

Basecalls performed using CASAVA version 1.8
For RNA-seq samples: SMART-seq2 reads were aligned to the mm9 genome assembly using tophat2 version 2.1.1, then replicates were merged together, and transcript abundance (FPKM) were calculated based on Refseq annotation using cufflinks version 2.0.2.
For Hi-C samples: sisHi-C sequencing reads were mapped, processed and iteratively corrected using HiC-Pro, a pipeline developed by Servant et al. Briefly, the read pairs were mapped to the mm9 reference genome in a two-step approach with bowtie2. Then the invalid read pairs including dangling end, self-circle ligation and duplicates were discarded. The genome was divided into bins of specific length to generate the contact maps. For global detection of contacts, a 100Kb bin size was used and a 40Kb bin size was used for examination of local domain level contacts. The raw contact counts are normalized with iterative correction.
For ChIP-seq samples: the paired-end ChIP reads were aligned with the parameters: -t –q –N 1 –L 25 –X 1000 --no-mixed --no-discordant to mm9 reference genome by Bowtie (version 2.2.2). All unmapped reads, reads with low mapping quality (MAPQ < 20) and PCR duplicates were removed.
For CUT&RUN samples:the pair-end CUT&RUN reads were aligned to mm9 reference genome with random chromosome cleaned by Bowtie (version 2.2.2) under the parameters –t –q –N 1 –L 25. All unmapped reads, reads with low mapping quality (MAPQ < 20) and PCR duplicates were removed.
Genome_build: mm9
Supplementary_files_format_and_content: The gene rpkm txt file contains FPKM value for all samples.

Submission date

Mar 17, 2020

Last update date

Jun 23, 2020

Contact name

Ke Zhang

E-mail(s)

zhangke16@mails.tsinghua.edu.cn

Organization name

Tsinghua university

Department

School of Life Sciences