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| Status |
Public on Dec 11, 2023 |
| Title |
Capture_enhancer_Mut_C6_rep1 |
| Sample type |
SRA |
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| Source name |
Cardiomyocyte
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Cardiomyocyte
day of differentiation: Day 38
genotype/variation: mutant
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| Growth protocol |
The iPSC lines were differentiated into cardiomyocytes using protocols adapted from Lian et al.29 and Zhang et al.30. Briefly, cells were seeded at a 1:5 ratio (approximately 200 000 cells/well) on 24 well plates coated 1:50 with reduced factor matrigel (matrigel basement membrane matrix growth factor reduced LDEV-free, Corning). They were then cultured in E8 medium until reaching 90% confluency (day 0). On day 0, -INS medium (RPMI 1640, Life Technologies Ltd, with N21-MAX insulin free media supplement, Bio-Techne) containing 8µM CHIR-99021 (Stratech Scientific Ltd), 10ng/ml recombinant Activin A (Bio-Techne), and 1:100 reduced factor matrigel were added for 24 hours. Following incubation (day 1), the medium was replaced with -INS medium and cells were cultured for a further 48 hours until day 3. They were then cultured in 1:1 old to fresh -INS medium supplemented with 5uM WNT inhibitor IWP-4 (Stemcell Technologies) until day 5, when the medium was replaced again with -INS medium for 48 hours. On day 7, the medium was switched to +INS medium (RPMI 1640, Life Technologies Ltd, with N21-MAX media supplement, Bio-Techne) and replaced every second day until more than 60% of cells were beating (day 12-15). They then received daily medium changes until harvesting (day 21 for ATAC sequencing and day 38 for RNA and C-capture experiments).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3C Libraries were generated according to the NuTi Capture-C protocol (Downes et al. 2020 in press). Briefly, cells were crosslinked with 2% formaldehyde (10 minutes, room temperature); quenched with cold glycine; washed in phosphate buffered saline; resuspended in cold lysis buffer (tris 10mM, NaCl 10mM, NP40 0.2%, complete proteinase inhibitor (Roche)) and snap frozen to -80. Cells were thawed on ice, washed in milliq dH2O and Dounce homogenised on ice (x 40 strokes). Cells were then resuspended with 0.25% SDS and restriction enzyme buffer and incubated at 37C for 1h at 1400rpm on a Comfort Thermomixer (Eppendorf) followed by a further incubation of 1h following the addition of triton X100 (final concentration 1.67%). An overnight digestion was performed using Dpn II (500U /ml (NEB) at 37C / 1400 rpm). The digested chromatin was ligated overnight (Fermentas HC Ligase final concentration 10U/ml) at 16 degrees at 1400 rpm on the Thermomixer. The samples were then decrosslinked overnight at 65C with Proteinase K (Roche) followed by a 30 min incubation at 37C with RNAse (Roche). Phenol/Chloroform extraction was then performed followed by an Ethanol precipitation and a wash with 70% Ethanol. Digestion efficiencys were assessed by gel electrophoresis (1% agarose) and RT-PCR (Taqman), which showed digestion efficiencies in excess of 70%. DNA content of the Dpn II 3C libraries were quantitated using a Qubit fluorometer (Life technologies)
5-10ug of each library was sheared using a Covaris S2 in milliq dH2O. Covaris settings used were: duty cycle 10%, Intensity 5, Cycles/burst 200, Time 6 cycles of 60seconds, Set Mode Frequency sweeping Temperature 4 to 7 degrees. Following shearing DNA was purified using AMPureXP beads (Agencourt) and DNA quality assessed on a Bioanalyser 2100 using a DNA High Sensitivity Chip (Agilent). DNA end repair and adapter ligation was performed using the NEB Next or NEB Ultra DNA sample preparation reagent kits, depending on the amount of DNA available, using the standard protocol. Biotinylated capture oligonucleotides were designed to the ends of the viewpoint fragments. Where possible 1-2ug of each adapter ligated library were hybridized with the biotinylated capture oligonucleotides, using the Nimblegen SeqCap reagents and an adapted protocol. The quality of the resultant captured library was assessed by Agilent tapestation or bioanalyser (D1000). The resulting libraries were sequenced using Illumina Nextseq 500 (150 bp paired-end reads)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
DpnII digested 3C library, enriched for fragments of interest by oligonucleotide pulldown
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| Data processing |
Library Strategy: NG Capture-C
Adaptor Removal: Trim Galore
Fragment reconstruction into a single read: Flash
In silico restriction enzyme digestion of FASTQ file (dpnIIPE.pl)
Alignment: Bowtie 2
Processing: Removal of PCR duplicates; Parsing of informative reads and mapping to restriction enzyme fragments (CCanalyser2.pl)
Processing: Combining data from multiple replicates (custom scripts)
Processing: Removal of ploidy regions and off target capture (custom scripts)
Genome_build: hg38
Supplementary_files_format_and_content: custom format (chr start stop interactions viewpoint)
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| Submission date |
Dec 14, 2020 |
| Last update date |
Dec 11, 2023 |
| Contact name |
Damien Downes |
| E-mail(s) |
damien.downes@ndcls.ox.ac.uk
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| Phone |
01865222374
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| Organization name |
The University of Oxford
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| Department |
MRC Weatherall Institute of Medicine
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| Street address |
John Radcliffe Hospital
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| City |
Headington |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DU |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE163172 |
Cardiomyocyte ST-wave depression indel characterisation (NuTi Capture-C) |
| GSE163174 |
Cardiomyocyte ST-wave depression indel characterisation |
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| Relations |
| BioSample |
SAMN17078291 |
| SRA |
SRX9683991 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4973799_Capture_enhancer_Mut_C6_rep1_interaction_counts.tab.gz |
85.8 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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