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Sample GSM3630268 Query DataSets for GSM3630268
Status Public on Apr 16, 2019
Title hDE_ScrgRNA_H3K27me3_rep1
Sample type SRA
 
Source name Definitive endoderm
Organism Homo sapiens
Characteristics cell_line: H1
assay: H3K27me3
treatment protocol: Scramble gRNA
chip antibody: Millipore, 07-451
Treatment protocol For perturbation experiments, H1-AAVS1-TetOn-dCas9-KRAB hESCs were transduced with individual gRNA lentiviral constructs and selected with puromycin for 72 hours. 48 hours prior to the start of differentiation, the selected cells were pooled and treated with 500 ng/mL doxycycline. The cells were then split for differentiation and differentiated along published protocols in the presence of 500 ng/mL doxycycline.
Growth protocol H1-AAVS1-TetOn-dCas9-KRAB hESCs were maintained in mTeSR1 on hES qualified matrigel-coated plates
Extracted molecule genomic DNA
Extraction protocol ATAC-seq samples were processed according to Buenrostro et. al with slight modifications. Briefly, approximately 50,000 cells were pelleted at 750 x g for 15 minutes at 4oC. After the addition of lysis buffer (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAl CA-630), nuclei were pelleted at 750 x g for 15 minutes at 4oC and the supernatent was discarded. Transposase reaction was performed at 37oC for 30 minutes. The purified, tagmented DNA was amplified for 9-10 cycles and size selected using AMPure XP beads (Beckman Coulter, A63881) as follows: 0.55X volume of 2X concentrated AMPure beads was added to the amplified DNA and incubated for 5 minutes at room temperature. The supernatant, containing low-molecular weight DNA, was collected in a separate tube by removing the beads using a magnetic rack. 1X volume of AMPure beads was added to the supernatant and incubated for 5 min at room temperature. The beads were washed twice with 75% ethanol and then air dried. The final purified library was eluted in EB buffer and sequenced (paired-end, 75-75) on a NextSeq500 or Hi-Seq2000 platform.
Total RNA was isolated using Trizol Reagent (Invitrogen, 15596-018) according to the manufacturer’s instructions. For quantitative PCR analysis, 1 μg of total RNA was reverse-transcribed using SuperScript III First-Strand Synthesis System (Thermo, 18080051).
Bulk RNA-sequencing library preparation of H1 hESC and differentiated hEND was performed following a published protocol {Zhang:2012kc}. Briefly, 4 μg of total RNA was depleted of ribosomal RNA using Ribo-Zero rRNA Removal Kit (Epicentre, MRZH116) and then converted to cDNA. Final sequencing libraries were generated using dUTP (Thermo, R0133) incorporation and uracil-N-glycosylase (NEB, M0280) treatment to generate strand-specific RNA-seq libraries. The final libraries were sequenced at paired-end on a Hi-Seq2000 platform.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description (no peak calls used)
Data processing Base calling: fastq files were obtained from Illumina Basespace under default parameters.
Bulk RNA-seq data utilized in atacTFAP analysis were trimmed, aligned, quantified, filtered, and tested for differential expression as follows.
Trimming: raw reads were trimmed 5bp from the head orientation and to 55 bp at the tail.
Alignment: reads were aligned with salmon version 0.8.2 to human reference transcriptome version GRCH37 using default parameters and setting the flags -l A --posBias --gcBias --seqBias.
Quantification: Transcript and gene expression levels were quantified using the salmon output and the txImport R package.
Filtering: we removed all genes with less than 3 (20th percentile) or more than 27337.16 (99.5th percetnile) reads across the dataset.
Differential expression analysis: performed using DESeq2. Genes with a minimal expression level of 5 RPKM in at least 2 samples and a FDR threshold of 0.01 were considered as differentially expressed.
ATAC-seq and ChIP-seq data underwent alignment, read extension, peak calling, IDR-based filtering, and differential analysis as follows.
Extension: For ChIP-seq only, reads were extended to 200 bp.
Alignment: reads were aligned to the human reference genome hg19 using Bowtie 2 version 2.3.2 using default parameters and filtering duplicate reads.
Peak calling and IDR: MACS2 in combination with the IDR framework was used for peak calling and detection of regions of genomic enrichment using an IDR cutoff of 0.1.
Differential analysis: we used the R package diffbind in combination with DESeq2 for all IDR based peak sets, requiring no overlap of peaks across conditions and using a DBA score based quantification.
Note: paired-end: R1 is a barcode
For RNA-seq, ATAC-seq, and ChIP-seq data visualization, IGV tools {Thorvaldsdottir:2013iw} was used to generate .tdf files.
Genome_build: hg19
Supplementary_files_format_and_content: Processed files are bed files containing peaks. Also included is one Excel file with total RNA results from hESC and hEND. Peaks were not used for K27me3 data, so no bed files were used in the analysis.
 
Submission date Feb 26, 2019
Last update date Apr 16, 2019
Contact name Rene Maehr
Organization name UMass Medical School
Department Program in Molecular Medicine
Lab Diabetes Center of Excellence
Street address 55 Lake Ave N
City Worcester
State/province MA
ZIP/Postal code 01604
Country USA
 
Platform ID GPL18573
Series (2)
GSE127201 Single-cell RNA sequencing-based CRISPRi screening resolves molecular drivers of early human endoderm development [set 2]
GSE127202 Single-cell RNA sequencing-based CRISPRi screening resolves molecular drivers of early human endoderm development
Relations
BioSample SAMN11022787
SRA SRX5431824

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record

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