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Status |
Public on Feb 03, 2020 |
Title |
[ChIP-seq, FLAG] MEF flagMyod1+Myt1l IP (rep1) |
Sample type |
SRA |
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Source name |
Mouse embryonic fibroblasts
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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)
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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.
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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.
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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.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NextSeq 500 |
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Description |
TGM_flagMyod+Myt1l_combinedinput.idrpeaks.0.1.merged.bed
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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)
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Submission date |
Feb 11, 2019 |
Last update date |
Feb 03, 2020 |
Contact name |
Qian Yi Lee |
E-mail(s) |
qianyi@stanford.edu
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Organization name |
Stanford University
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Department |
Bioengineering
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Lab |
Wernig Lab
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Street address |
265 Campus Drive
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City |
Stanford |
State/province |
CA |
ZIP/Postal code |
94305 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (2) |
GSE126410 |
Pro-neuronal activity of Myod1 due to promiscuous binding [FLAG ChIP-seq] |
GSE126414 |
Pro-neuronal activity of Myod1 due to promiscuous binding |
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Relations |
BioSample |
SAMN10911903 |
SRA |
SRX5360409 |
Supplementary data files not provided |
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Raw data are available in SRA |
Processed data are available on Series record |
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