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Sample GSM3581647 Query DataSets for GSM3581647
Status Public on Jun 19, 2019
Title P33T-PP_PDX1_Lane1_CTTGTA
Sample type SRA
 
Source name PDX1P33T/+ iPS derived pancreatic precursor cells
Organism Homo sapiens
Characteristics assay: PDX1
lane: Lane1
antibody: Goat anit PDX1 (Christopher V. E., Wright)
Treatment protocol PDX1P33T/+ iPSCs plated on 1:100 diluted Matrigel were first rinsed with 1× DPBS without Mg2+ and Ca2+ (Invitrogen) and then cultured in RPMI 1640 medium (Invitrogen) further supplemented with 1.2 g/L sodium bicarbonate (Sigma), 0.2% ESC-qualified FBS (Life Technologies), 100 ng/mL Activin-A (R&D Systems), and 20 ng/mL of Wnt3A (R&D Systems) for day 1 only. For the next 2 days, cells were cultured in RPMI with 0.5% FBS, 1.2g/L sodium bicarbonate and 100 ng/mL Activin-A ; Cells were rinsed with 1× DPBS (without Mg2+ and Ca2+) once and then exposed to DMEM-F12 (Life Technologies) medium further supplemented with 2 g/L sodium bicarbonate, 2% FBS and 50 ng/mL of FGF7 (R&D Systems) for 3 days; Cultures were maintained for 4 days in DMEM-HG medium (Life Technologies) supplemented with 0.25 µM SANT-1 (Sigma-Aldrich), 2 µM retinoic acid (RA; Sigma-Aldrich), 100 ng/mL of Noggin (R&D Systems), and 1% B27 (Invitrogen).
Growth protocol PDX1P33T/+ iPSCs were cultured on 1:100 diluted Matrigel (BD Biosciences) in mTeSR™1 medium (Stem Cell Technologies). At ~70–80% confluency, cultures were rinsed with 1× DPBS without Mg2+ and Ca2+ (Invitrogen) followed by incubation with 1× TrypLE Select Enzyme (Life Technology) for 3–5 min at 37 °C. Single cells were rinsed with mTeSR™1 medium, and spun at 1,000 rpm for 3 min. The resulting cell pellet was resuspended in mTeSR™1 medium supplemented with 10 μM Y-27632 (Sigma-Aldrich) and the single cell suspension was seeded at ~1.0 × 105 cells/cm² on Matrigel-coated surfaces. Cultures were fed every day with mTeSR™1 medium and differentiation was initiated 48 h following seeding, resulting in ~90% starting confluency.
Extracted molecule genomic DNA
Extraction protocol PDX1P33T/+ PP cells (2 × 106 cells) were cross-linked in 1% formaldehyde in culture medium for 10 min at room temperature. The cross-linking reaction was stopped by the addition of glycine to a final concentration of 125 mM. For chromatin fragmentation, cells were resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS) and sonicated in a Covaris S220 sonicator with a duty cycle of 2%, a peak incident power of 105 W and 200 cycles per burst for 20 min. The fragmented chromatin was diluted 1:5 in IP-Buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-Desoxycholate, 140 mM NaCl, H2O, Protease Inhibitors) and directly used for immunoprecipitation. For PDX1 ChIP, 60% of the chromatin (equivalent of 1.2 × 106 cells) was processed in two parallel ChIPs using magnetic beads, preloaded with 3 µl goat anti PDX1 antibody (kindly provided by C. Wright), for each ChIP. For H3K27ac ChIP, 40% of the chromatin (equivalent 0.4 × 106 cells) was processed in three parallel IPs using magnetic beads, preloaded with 3 µg anti H3K27ac antibody (Diagenode, pAb-174-050). For all ChIPs, antibody incubation was performed at 4 °C for 5 h. Beads were then washed 5 times using (1.) IP-Buffer, (2.) Washing-Buffer 1 (500 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% NP-40), (3.) Washing-Buffer 2 (250 mM LiCl, 50 mM Tris-HCl (pH 8.0), 0.5% Na-Deoxycholate, 1% NP-40) and (4. & 5.) Washing-Buffer 3 (10 mM Tris-HCl (pH 8.0), 10 mM EDTA). Subsequently, protein-DNA complexes were eluted from the beads in Elution-Buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS) at 65 °C for 20 min. Cross-links were reversed at 65 °C overnight and DNA was purified by ethanol precipitation.
Libraries were constructed using the MicroPlex kit (Diagenode, C05010010). Library construction, purification and size selection using AMPureXP (Beckman Coulter) were performed according to the MicroPlex kit instruction manual (version 4 / 03.09.14)
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 1500
 
Description P33T_PDX1_Multi-Condidtion_GEM_events.bed
Data processing Fastq files were preprocessed with Trimmomatic (0.35) with parameters CROP:50 ILLUMINACLIP:path/to/adapter.fa:2:30:10:1 LEADING:0 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36.
Preprocessed Fastq files were aligned to hg19 genome using bowtie2 (2.2.6) with very-sensitive option.
SAM files were converted to BAM and duplicate reads were removed using samtools (1.3).
Deduplicated BAM files from PDX1P33T/+ PDX1 and H3H27ac were subsampled to match the read depth of the corresponding experiments in XM001 cells (GSE106949)
Binding sites for PDX1 were then called using GEM (3.2) in multicondition mode using data from XM001 (GSE106949) and PDX1P33T/+ (this study) PPs. Bindingsites were filtered, after visual inspection, using a Q-value cut-off of 10^-4 (XM001) and 10^-4.05 (PDX1P33T/+). Overlapping regions were merged and peaks overlapping blacklist regions were removed using bedtools (2.26.0).
Regions of H3K27ac enrichment were called using HOMER (4.10) on the merged data from XM001 (GSE106949) and PDX1P33T/+ (this study) cells using parameters -style histone -size 500 -minDist 2000. Regions overlapping blacklist-regions were discarded.
Regions and binding sites within 20 kb of a TSS or within a gene body were annotated with the respective gene using bedtools (2.26.0).
Genome_build: hg19
Supplementary_files_format_and_content: BED file containing peak locations
 
Submission date Jan 28, 2019
Last update date Jun 19, 2019
Contact name Heiko Lickert
E-mail(s) heiko.lickert@helmholtz-munich.de
Organization name Helmholtz Zentrum München German Research Center for Environmental Health
Department Institute of Diabetes and Regeneration Research
Street address Ingolstaedter Landstraße 1
City Neuherberg
ZIP/Postal code 85764
Country Germany
 
Platform ID GPL18460
Series (2)
GSE125768 Point mutations in the PDX1 transactivation domain impair human β-cell development and function (ChIP-Seq)
GSE125770 Point mutations in the PDX1 transactivation domain impair human β-cell development and function
Relations
BioSample SAMN10833646
SRA SRX5300515

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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