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Sample GSM3598024

Query DataSets for GSM3598024

Status

Public on Feb 03, 2020

Title

[ChIP-seq, FLAG] MEF flagMyod1+Myt1l IP (rep1)

Sample type

SRA

Source name

Mouse embryonic fibroblasts

Organism

Mus musculus

Characteristics

cell type: Mouse embryonic fibroblasts
transgenes: flagMyod1 + Myt1l + rtTA
days post transgene induction: 2 days
chip antibody: FLAG-conjugated beads (Sigma F2426)

Treatment protocol

P3 MEFs were co-infected with reverse tetracycline transactivator (rtTA) and doxycycline (dox) inducible Ascl1 or Myod1 overnight and then cultured in MEF media (DMEM; Invitrogen) containing 10% cosmic calf serum (CCS; Hyclone), beta-mercaptoethanol (Sigma), non-essential amino acids, sodium pyruvate and penicillin/streptomycin (all from Invitrogen) supplemented with 2 µg/ml dox (Sigma) for 48h.

Growth protocol

Homozygous TauEGFP knock-in mice were bred with C57B/6 mice (Charles River) to generate heterozygous embryos. Heterozygous TauEGFP knock-in TauEGFP mouse embryonic fibroblasts (MEFs) were then isolated from E13.5 embryos.

Extracted molecule

genomic DNA

Extraction protocol

FLAG-tagged TFs were immunoprecipitated using FLAG M2 antibody conjugated beads (Sigma F2426). Approximately 60-100 million cells were used per replicate. Beads (~100uL slurry per sample) were washed thrice with IP dilution buffer (1% Triton X-100, 2mM EDTA pH8.0, 20mM Tris-HCl pH8.0, 150mM NaCl, 1mM DTT, 100uM PMSF), blocked overnight in IP dilution buffer with 0.1% BSA and 0.06% sheared salmon sperm DNA (Thermo Fisher; AM9680), then washed again three times with IP dilution buffer before use. Nuclei were isolated by incubating with cell lysis buffer (5mM HEPES pH7.9, 85mM KCl, 0.5% NP40, 100uM PMSF, protease inhibitors from Roche) for 10min on ice and centrifuged at 5000rpm for 5min at 4°C, then lysed with nuclear lysis buffer (50mM Tris-HCl pH8.0, 10mM EDTA pH8.0, 1% SDS, 100uM PMSF, protease inhibitors) for 10min on ice. Chromatin was sheared using either the Bioruptor (Diagenode) or Covaris sonicator until DNA was fragmented to 200-500bp. Sheared chromatin was diluted using 3X volume of IP dilution buffer and pre-cleared for at least 4hr at 4°C using IgG beads (Sigma A0919). 1% of pre-cleared chromatin was kept as input, and the remainder was incubated with FLAG beads overnight at 4°C. Beads were washed 8 times with IP wash buffer (20mM Tris-HCl pH8.0, 2mM EDTA pH8.0, 250mM NaCl, 1% NP40, 0.05% SDS, 100uM PMSF) and once with TE buffer with 100uM PMSF. Beads and 1% input samples were reverse cross-linked overnight in IP elution buffer (50mM NaHCO3, 1% SDS) at 65°C. Isolated DNA was RNase treated for 30min at 37°C, purified using QIAquick PCR purification columns (Qiagen) and eluted in DEPC water.
ChIP-seq libraries were prepared using the NEBNext library prep kit (NEB; E6240) following the supplier’s protocols with slight modifications. Adaptor ligation was performed using regular T4 ligase and ligase buffer. Size selection (200-500bp) was performing after adaptor ligation and PCR enrichment using 2% low-melt agarose gel (Lonza; 50080), and DNA was purified using the QiaQuick gel extraction columns from Qiagen.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

TGM_flagMyod+Myt1l_combinedinput.idrpeaks.0.1.merged.bed

Data processing

Reads were aligned to the mm10 reference sequence using Bowtie 2.1.0 with an additional -5 10 parameter.
Peak calling was performed using MACS 2.1.1 .
For experiments with two replicates, reproducible peaks were selected using IDR 2.0.2. with cutoff of idr ≤ 0.1 using the recommended pipeline for MACS2. For experiments with ≥ 3 replicates, pairwise IDR was performed and reproducible peaks (idr ≤ 0.1) for each pair were merged into a single peak list using Diffbind 1.16.3 with minOverlap = number of replicates.
Genome_build: mm10
Supplementary_files_format_and_content: Tab delimited file of merged peaks (chr, start, stop, name, p-value, strand)

Submission date

Feb 11, 2019

Last update date

Feb 03, 2020

Contact name

Qian Yi Lee

E-mail(s)

qianyi@stanford.edu

Organization name

Stanford University

Department

Bioengineering