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| Status |
Public on May 30, 2023 |
| Title |
WT, 3h dTAG, rep2, TWIST1 |
| Sample type |
SRA |
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| Source name |
H9
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell type: Human cranial neural crest cell
cell line: H9
genotype: WT
clone: --
treatment: dTAGV-1 (500 nM)
time: 3h
spike-in cell_type: Mouse cranial neural crest cell
spike-in cell_line: O9-1
antibody: TWIST1 (Abcam, #ab50887)
reference genome_for_alignment: hg38 + mm39
reference genome_for_processed_files: hg38
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| Treatment protocol |
DMSO or dTAGV-1 (500 nM) were added by dilution into media before changing media. HEK293 cells were transfected with Lipofectamine 2000 (Invitrogen, 11668019) at a ratio of 2.8 ul lipofectamine per ug of DNA, diluted in Opti-MEM. Cells were transfected with 2.5 ug DNA per well of a 6-well plate or 15 ug DNA per 10-cm plate 1-2 days after seeding, when they reached 70-90% confluency. Media was replaced 4-6h after transfection, and then cells were harvested for Western blot or chromatin immunoprecipitation at 24 h after transfection.
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| Growth protocol |
H9 cells (WiCell, WA09, RRID:CVCL_9773) were cultured in feeder-free conditions, in mTeSR1 medium (Stem Cell Technologies, 85850) on Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, 356231) and passaged using ReLeSR (Stem Cell Technologies, 05872) every 4-6 days. Cells were switched to mTeSR Plus medium (Stem Cell Technologies, 100-0276) prior to and during genome editing and clonal expansion, but switched back to mTeSR1 before differentiation to CNCC. hESCs were differentiated to CNCC as previously described. Briefly, hESC colonies were partially detached from the plate with collagenase IV (Gibco, 17104019) in Knockout DMEM medium (Gibco, 10829018) for 30-60 min and scraped to break up large colonies, and then cultured in Neural Crest Differentiation Medium (50%-50% v/v mixture of DMEM/F12 1:1 medium with L-glutamine, without HEPES (Cytiva, SH30271.FS) and Neurobasal medium (Gibco, 21103049) with 0.5x N2 NeuroPlex (Gemini Bio, 400-163) and Gem21 NeuroPlex (Gemini Bio, 400-160) supplements and GlutaMAX (Gibco, 35050061), and 1x antibiotic/antimycotic, and 20 ng/ml EGF (Peprotech, AF-100-15), 20 ng/ml bFGF (Peprotech, 100-18B), and 5 ug/ml bovine insulin (Gemini Bio, 700-112P)) for 11 days in bacterial-grade petri dishes, changing the plate to prevent attachment for 4 days and then leaving the cells unfed for two days to allow attachment, and then fed as needed at least every other day. At day 11, cells (now called ‘early hCNCC’) were harvested by treatment with Accutase (Sigma-Aldrich, A6964-100ML), strained to remove residual neuroectodermal spheres, and plated onto plates coated with 7.5 ug/ml human fibronectin (Millipore, FC010-10MG) and cultured in Neural Crest Maintenance Medium (Neural Crest Differentiation Medium with bovine insulin replaced by 1 mg/ml BSA (Gemini Bio, 700-104P)). These hCNCC were then passaged every 2-3 days upon reaching confluency, with cells in the third or subsequent passages defined as ‘late hCNCC’ and cultured with added 50 pg/ml BMP2 (Peprotech, 120-02) and 3 uM CHIR-99021 (Selleck, S2924). dTAGV-1 (Tocris, 6914/5) was dissolved in DMSO at 5 mM and then diluted to 250 uM in 60% DMSO/40% water (v/v) before dilution to 500 nM for acute depletions (up to 1 day) or diluted directly from the 5 mM stock for long-term depletions. RS4;11 cells (ATCC, CRL-1873, RRID:CVCL_0093) were cultured in RPMI-1640 medium (Gibco, 11875093) supplemented with 10% v/v FBS and 1x antibiotic/antimycotic. HEK293 cells (ATCC, CRL-1573, RRID:CVCL_0045) and 293FT cells (Invitrogen, R70007, RRID:CVCL_6911) were cultured in DMEM high glucose medium with sodium pyruvate and L-glutamine, supplemented with 10% v/v FBS and 1x GlutaMAX, non-essential amino acids, and antibiotic/antimycotic. O9-1 cells (Millipore, SCC049, RRID:CVCL_GS42) used for spike-in controls for ChIPs of TWIST1 depletions were cultured in Complete ES Cell Medium with 15% FBS (Millipore, ES-101-B), 25 ng/ml bFGF, and mLIF (Millipore, ESG1107).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells (about 1 confluent 10-cm plate or ~10-20 million cells) were crosslinked with 1% methanol-free formaldehyde (Pierce, 28908) in PBS for 10 min at room temperature and then quenched by adding 2.5 M glycine to 125 mM final concentration and incubating for 10 min. Cells were washed in PBS with 0.001% v/v Triton X-100, harvested by scraping, and collected by centrifugation for 5 min at 4°C. Cells were washed with PBS and flash frozen for storage at -80°C. Cell pellets were later thawed on ice for 30 min, and then sequentially resuspended in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Igepal CA-630, 0.25% Triton X-100, 1x cOmplete EDTA-free protease inhibitor cocktail (PIC), 1 mM PMSF), lysis buffer 2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x cOmplete EDTA-free protease inhibitor cocktail, 1 mM PMSF), and lysis buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1x PIC, 1 mM PMSF), with 10 min incubations in each buffer, with rotation. Lysates were sonicated for 10-15 cycles of 30s ON/30s OFF on high power using the Bioruptor Plus (Diagenode), then diluted in additional lysis buffer 3 and clarified by centrifugation for 10 min at max speed at 4°C. Triton X-100 was added to 1%, and a small aliquot was used to extract DNA to check chromatin yield and size distribution, by dilution in elution buffer (1% w/v SDS and 100 mM NaHCO3) and incubation with 200 mM NaCl and RNase A (Thermo, EN0531) at 65°C for 1 h, then proteinase K (Thermo, EO0491) at 65°C for 1 h, and clean up with the ChIP DNA Clean & Concentrator-5 kit (Zymo, D5205). DNA was quantified by Qubit dsDNA high sensitivity kit, and the remaining chromatin was then normalized for immunoprecipitations. For TWIST1 acute depletions, chromatin from O9-1 mouse CNCCs were added prior to ChIP at ~10% of the total chromatin as a spike-in control. For H3K27ac, 5 ug of antibody was used per ChIP; for TFs, 9 ug of antibody was used per ChIP, except for dissected mouse embryos where 4.5 ug was used in half of the total ChIP volume. ChIPs were incubated overnight, then incubated for 4-6h with 100 ul Dynabeads Protein A (Invitrogen, 10002D) or Protein G (Invitrogen, 10004D) prewashed with 0.1% w/v BSA in PBS, then washed 5x with RIPA wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% Igepal CA-630, 0.7% w/v sodium deoxycholate), once with 50 mM Tris-HCl pH 8, 10 mM EDTA, 50 mM NaCl, and eluted in elution buffer at 65°C for 30 min. Eluate was then reverse crosslinked and treated with RNase A and proteinase K, and then DNA was extracted with the ChIP DNA Clean & Concentrator-5 kit.
Libraries were prepared using the NEBNext Ultra II DNA kit (New England Biolabs, E7645S) using up to 50 ng of input or ChIP DNA, with ~4-8 cycles of amplification, with no pre-PCR size selection but a post-PCR double-sided 0.5x/0.9x Ampure XP bead clean-up.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were trimmed of Truseq adapter sequences and low-quality bases (-Q 10) using skewer v0.2.2.
Trimmed reads were mapped to the hg38 analysis set (human), mm39 (mouse), or a combined reference genome using Bowtie2 v2.4.1 with the options --very-sensitive -X 2000.
Reads were deduplicated with samtools v1.10 markdup and uniquely mapped reads (-q 20) mapped to the main chromosomes (excluding mitochondria and unplaced contigs) were retained using samtools view.
MACS2 v2.2.7.1 was used to call peaks using the IgG sample as a negative control, with options -f BAMPE --nomodel --keep-dup all --call-summits -g hs (human) or mm (mouse) with the -B option for H3K27ac.
bigWig tracks were generated with deeptools v3.5.0 bamCoverage -bs 10 --normalizeUsing RPGC --samFlagInclude 64 --samFlagExclude 8 --extendReads with 1h dTAG samples normalized by the fraction of reads mapping to the E. coli genome.
H3K27ac ChIP reads were counted over merged ATAC peaks using deeptools v3.5.0 multiBamSummary with options -e --outRawCounts.
Assembly: hg38 or mm39
Supplementary files format and content: bigWig, narrowPeak (except for input samples, H3K27ac ChIPs, and V5 ChIPs of dTAG-treated samples) for each sample, and tab-delimited text (.txt) files with read counts of H3K27ac ChIPs over merged ATAC peaks
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| Submission date |
Apr 21, 2023 |
| Last update date |
May 30, 2023 |
| Contact name |
Seungsoo Kim |
| Organization name |
Stanford University
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| Department |
Chemical and Systems Biology
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| Lab |
Joanna Wysocka
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| Street address |
265 Campus Dr
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL25526 |
| Series (2) |
| GSE230316 |
DNA-guided transcription factor cooperativity shapes face and limb mesenchyme [ChIP-seq] |
| GSE230319 |
DNA-guided transcription factor cooperativity shapes face and limb mesenchyme |
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| Relations |
| BioSample |
SAMN34296360 |
| SRA |
SRX20052982 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7213632_WT_dTAG-3h_r2_TWIST1.bin10.bw |
96.7 Mb |
(ftp)(http) |
BW |
| GSM7213632_WT_dTAG-3h_r2_TWIST1_peaks.narrowPeak.gz |
1.3 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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