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| Status |
Public on Apr 24, 2022 |
| Title |
Ctr3_LCL_CapC |
| Sample type |
SRA |
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| Source name |
human lymphoblastoid cell line
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| Organism |
Homo sapiens |
| Characteristics |
phenotype: unaffected
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| Growth protocol |
LCLs derived from ATR-X patients and healthy controls were incubated with RPMI medium supplemented with 15% FCS, 1% PenStrep and 1% glutamine at 37°C and 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3C Libraries were generated according to the NG Capture-C protocol (Davies et al. 2016). 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: hg19
Supplementary_files_format_and_content: custom format (chr start stop interactions viewpoint)
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| Submission date |
Jan 09, 2022 |
| Last update date |
Apr 24, 2022 |
| Contact name |
Julia Truch |
| Organization name |
University of Oxford
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| Department |
RDM - WIMM
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| Lab |
GIBBONS/HIGGS
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| Street address |
John Radcliffe Hospital, Headley Way, Headington
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| City |
Oxford |
| ZIP/Postal code |
Oxford OX3 9DS |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (1) |
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| Relations |
| BioSample |
SAMN24776744 |
| SRA |
SRX13684807 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5792008_Ctr3_LCL_CapC_interaction_counts.tab.gz |
29.4 Mb |
(ftp)(http) |
TAB |
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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