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| Status |
Public on Apr 18, 2022 |
| Title |
Replicate 2; Control Day 5 differentiated hiPSCs |
| Sample type |
SRA |
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| Source name |
WTC CRISPRi GCaMP hiPSC (RRID CVCL_VM38)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: WTC CRISPRi GCaMP hiPSC (RRID CVCL_VM38)
cell type: Day 5 differentiated human induced pluripotent stem cells
genotype: WT
treatment: cardiac differentiation
time: Day 5
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| Treatment protocol |
An established monolayer-based cardiac directed differentiation protocol was modified and used to differentiate WTC CRISPRi GCaMP hiPSCs. On day -1 hiPSCs were dissociated using 0.5% EDTA and plated at a density of 1.8x105 cells/cm2 on Vitronectin XF coated plates and cultured overnight in mTeSR media at 37°C. To initiate differentiation on day 0 cells were washed with PBS, then fed with RPMI media (ThermoFisher, Cat.#11875119) containing 3mM Wnt-agonist CHIR99021 (Stem Cell Technologies, Cat.#72054), 500mg/mL BSA (Sigma Aldrich, Cat.#A9418), and 213mg/mL ascorbic acid (Sigma Aldrich, Cat.#A8960). After 3 days of culture, the media was replaced with RPMI containing 500mg/mL BSA, 213mg/mL ascorbic acid, and 5mM Wnt-antagonist XAV-939 (Stem Cell Technologies, Cat.#72674). On day 5, the media was exchanged for RPMI containing 500mg/mL BSA, and 213mg/mL ascorbic acid without supplemental cytokines.
Cells were treated with 1uM MS275 and 1uM BRD4884 for 48 hours prior to sample collection on Day 5 of differentiation
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| Growth protocol |
WTC CRISPRi GCaMP hiPSCs were maintained on 5μg/mL Vitronectin XF (Stem Cell Technologies, 07180) coated plates in mTESR1 media (Stem Cell Technologies, 05850) treated with 1% penicillin/streptomycin at 37°C with a 5% CO2 incubator
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Omni-ATACseq sequencing libraries were generated from Day 5 cells undergoing hiPSC cardiac-directed differentiation using an adapted published workflow (Corces et al., 2017). Briefly day 5 cells were harvested and 15,000 cells were pelleted at 500 x g for 5 min at 4oC and washed once in 50 µl cold PBS. The cell suspension was then pelleted again at 500 g for 5 min at 4oC. For cell lysis, the pellets were resuspended in 50 µl cold ATAC-Resuspension Buffer (10 mM Tris-HCl pH 7.4 (Thermo Scientific, 10 mM NaCl (Sigma), and 3 mM MgCl2 (Sigma) in nuclease-free water (Thermo Scientific) containing 0.1% (v/v) NP40 (Sigma), 0.1% (v/v) Tween-20 (Sigma), and 0.01% (v/v) Digitonin (Promega) by gently pipetting up and down three times. After cell lysis, 1 ml of cold ATAC-Resuspension Buffer was added and mixed by inverting the tubes. The samples were then centrifuged at 500 g for 10 min at 4oC and supernatant is carefully removed away from the pellets. For the transposition reaction, each pellet was resuspended in 12.5 µl of transposition mix (6.25 µl 2x TD buffer (Illumina), 0.625 µl Tn5 (Illumina), 4.125 µl PBS, 0.125 µl 1% (v/v) Digitonin (Promega), 0.125 µl 10% (v/v) Tween-20 (Sigma), 1.25 µl nuclease-free water (Thermo Scientific) by gently pipetting up and down six times. The samples were then incubated at 37oC for 30 min in a thermomixer shaking at 300 rpm, followed by immediate purification using a DNA Clean & Concentrator-5 Kit (Zymo, Cat.# D4013) according to the manufacturer’s specification, except for the final elution volume, which was 21 µl. A PCR reaction volume of 50 µl was then set up using 20 µl of the purified DNA, 25 µl of the NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs) and 1.25 mM of the ATACseq oligos published previously (Buenrostro et al., 2013). The DNA samples were amplified with the following parameters: 72oC for 5 min, 98oC for 30 s and 11 cycles of 98oC for 10 s, 63oC for 30 s and 72oC for 30 s.
The omni-ATAC libraries were then purified using the DNA Clean & Concentrator-5 Kit (Zymo) according to the manufacturer’s specification and eluted in 20 µl nuclease-free water (Thermo Scientific). The libraries were quantified with Qubit dsDNA HS Assay Kit (Invitrogen) and assessed for library size distribution using an Agilent Bioanalyzer.The libraries were then pooled and sequenced in 2x100 cycle paired-end mode on an Illumina Novaseq instrument.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
ATAC-seq data processing and alignment was completed using the Harvard pipeline (https://informatics.fas.harvard.edu/atac-seq-guidelines.html). All fastq files initial quality was assessed using FastQC v0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Bowtie2 (Langmead and Salzberg, 2012) was used to aligned reads to the GRC38_no_alt genome bowtie 2 index for alignment (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml), using “-p 8, –very-sensitive” options. Picard tools (http://broadinstitute.githyb.io/picard/) was used to mark and remove duplicates using the MarkDuplicates tool with default options. Samtools (Li et al., 2009) was used to sort and obtain uniquely mapped reads suing “-b -q 10” options ato remove reads from the mitochondrial chromosome. This resulted in the final aligned, deduplicated BAM files that were used for downstream analysis.
Peak calling was performed to ensure high quality fixed-width peaks in accordance with Corces et al.(Corces et al., 2018). For each sample, peak calling was performed using the MACS2 callpeak command with the following parameters “-g mm –shift -100 –extsize 200 –nomodel –call-summits –nolambda –keep-dup all -p 0.01” (Feng et al., 2012; Zhang et al., 2008). Then, peak summits were extended by 250 bp on both sides to a final width of 501 bp. Peaks were filtered for GRC38 blacklisted regions (Amemiya et al., 2019) (https://www.encodeproject.org/annotations/ENCSR636HFF/).
As in Corces et al. 2020 per sample overlapping peaks were handled using an iterative removal approach. That is in case of two overlapping peaks the most significant peak is kept and the other removed. This process is performed in an iterative manner, so all peaks have been kept or removed based on their overlap and significance score. This method results in a set of fixed-width peaks per sample.
As in Corces et al. (Corces et al., 2018) per sample overlapping peaks were handled using an iterative removal approach. That is in case of two overlapping peaks the most significant peak is kept and the other removed. This process is performed in an iterative manner, so all peaks have been kept or removed based on their overlap and significance score. This method results in a set of fixed-width peaks per sample. Due to samples with higher sequencing depth and quality yield a higher number of MACS2 original number of peaks we normalized their significance peak scores “(-log10(p-value))” to a score per million value by dividing each individual peak score by the sum of all of the peak scores in the given sample divided by 1 million (Corces et al., 2018). Thus, scores per million allow the direct comparison of peaks across biological replicates. Next, we complied a peak set across conditions. For that we generated a cumulative peak set across samples and performed the same iterative approach as mentioned above. Only peaks with a score per million value >=3 observed in at least two samples (minimal overlap 50%) were further considered. This resulted in a set of fixed-width, reproducible, high-quality peaks across conditions of 83,554 peaks, each with 501 bp in width.
To assess differential accessibility between conditions the across-conditions ATAC-seq peaks was converted to gtf format. The number of reads falling within these regions per sample were calculated using the featurecounts function within the Subread package (version2.0.1) (Liao et al., 2014). Next, a count matrix containing the samples of interest was inputted into the DESeq2 package (Love et al., 2014), where only peaks with at least 5 counts per million in at least 2 samples were further processed to identify differentially accessible regions between conditions.
Assembly: hg38
Supplementary files format and content: tab-delimited text files
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| Submission date |
Apr 14, 2022 |
| Last update date |
Apr 19, 2022 |
| Contact name |
Nathan J. Palpant |
| Organization name |
University of Queensland
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| Street address |
306 Carmody Rd
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| City |
Brisbane |
| State/province |
QLD |
| ZIP/Postal code |
4067 |
| Country |
Australia |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE200808 |
Temporal perturbation of histone deacetylase activity reveals a requirement for HDACs 1-3 in mes-endoderm cell differentiation |
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| Relations |
| BioSample |
SAMN27585093 |
| SRA |
SRX14857948 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6043951_C2_2_S8.bw |
768.4 Mb |
(ftp)(http) |
BW |
| GSM6043951_C2_2_S8.narrowPeak.txt.gz |
4.0 Mb |
(ftp)(http) |
TXT |
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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