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Sample GSM3854036

Query DataSets for GSM3854036

Status

Public on Apr 10, 2020

Title

Cfp1_KO_input_rep2

Sample type

SRA

Source name

th17 cells

Organism

Mus musculus

Characteristics

chip antibody: none
cell type: naive CD4-derived th17 cells

Growth protocol

Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) were purified by FACS Aria II flow cytometer or sorted by Mouse CD4 Naïve T cell Enrichment Kit (Invitrogen, # 8804-6824-74). Naive CD4+T cells were cultured with irradiated (30 Gy) APC sorted from spleen at a ratio of 1:3, and were activated with 2µg/ml anti-CD3 and 3 µg/ml anti-CD28 in 48-well plate (5×105 T cells/well). T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, sodium pyruvate, penicillin/streptomycin, and 2-mercaptoethanol.For Th17 cell differentiation, culture medium was supplemented with IL-6 (20 ng/mL), TGF-β1 (5 ng/mL), anti-IL-4 (10 ng/mL), anti-IL-12(10 ng/mL), anti-IFNγ (10 ng/mL).

Extracted molecule

genomic DNA

Extraction protocol

ChIP assays were performed according to the manufacturer’s instructions with modifications using the ChIP-IT kit (Active Motif, USA). In brief, the Th17 cells were fixed with 1% formaldehyde, and then the crosslinked chromatin was sonicated in a 4°C water bath using a Bioruptor Pico sonicator (Diagenode) to obtain DNA fragments between 150 and 500 bp in size. For Cxxc1 ChIP-seq, 5×106 Th17 cells and 6 µg of Cxxc1 antibody were used for each sample. For H3K4me3 ChIP-seq, 3×106 Th 17 cells and 4 µg of H3K4me3 antibody were used for each sample.
The immunoprecipitated DNA was purified and subjected to sequencing library preparation using a VAHTSTM Universal DNA Library Prep Kit for Illumina® V2 (Vazyme Biotech Co., Ltd) according to the manufacturer’s protocol. The DNA libraries were then sequenced with an Illumina HiSeq X ten system at Veritas Genetics in Hangzhou.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

HiSeq X Ten

Description

ChIP-seq of mus musculus

Data processing

Peakcalling performed using MACS2 version 2.1.1 with parameters -q 0.05 --nomodel --extsize 300 -B -SPMR
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using bowtie2 v2.2.6
Genome_build: mm10

Submission date

Jun 05, 2019

Last update date

Apr 11, 2020

Contact name

lie wang

E-mail(s)

wanglie@zju.edu.cn

Organization name

zhejiang universiry

Department

immunology