Sample 3
Homo sapiens
Protocols: SGBS cells are referenced here: https://doi.org/10.1038/sj.ijo.0801520. Cells were collected and single-cell suspension was made. 10^7 cells were aliquoted in 10ml media. Cells were collected in DMEM 4.5g/mL. 270ul 37% formaldehyde was added and incubated for 10mins at RT tumbling, then quenched by adding 1.5ml 1M cold glycine (4C) giving a 12ml total. Sample was tumbled for 5min after glycine addition. After 5min centrifuge at 1000rpm and 4C the supernatant was removed. The pellet was washed in 10ml cold PBS (4C), then centrifuged for 5min at 1000rpm and 4C, then PBS was removed. 35uL of fresh 200x protease inhibitor cocktail were added to 7mL of prepared 3C lysis buffer and mixed thoroughly. The lysis buffer was made by combing 10mM Tris pH8 (500ul 1M), 10 mM NaCl (125ul 4M), 0.2% NP-40 (Igepal) (100ul 100%), and 49.25ml Milli Q in a 50ml Falcon and rotated at 4C to mix (stored at 4C). Cell pellet was resuspended in 5ml cold lysis buffer and incubated for 20min on ice, samples they were placed on an platform shaker to rock lightly during incubation, then centrifuged for 5min at 1800rpm and 4C, then the lysis buffer removed. Pellet was resuspended in 1ml lysis buffer and transferred to 1.5ml Eppendorf prior to snap freezing (ethanol/dry ice or liquid Nitrogen). Cells could be thawed again for digestion at this point or stored at -80C. The initial 3C libraries were generated following a previously published protocol (Hughes et al., Nature genetics, 2014). The DNA was digested using DpnII, then re-ligated together using T4 DNA ligase, and finally isolated by phenol/chloroform extraction. The 3C libraries were sonicated to an average size of 300bp. Agilent SureSelect library capture protocol was then applied with capture probes targeting both ends of DpnII restriction fragments that overlap promoters of protein-coding, noncoding, antisense, snRNA, miRNA, snoRNA and lincRNA transcripts, totaling 36,691 RNA baited fragments. The bait list is provided in the file baits_1frag.bed.
ENA-FIRST-PUBLIC
2019-04-30T04:02:46Z
ENA-LAST-UPDATE
2018-12-19T14:50:25Z
External Id
SAMEA5190630
INSDC center name
The Children's Hospital of Philadelphia
INSDC first public
2019-04-30T04:02:46Z
INSDC last update
2018-12-19T14:50:25Z
INSDC status
public
Submitter Id
E-MTAB-7550:Sample 3
broker name
ArrayExpress
cell line
SGBS
cell type
stromal cell
common name
human
developmental stage
infant
disease
Simpson-Golabi-Behmel syndrome
organism part
subcutaneous adipose tissue
sample name
E-MTAB-7550:Sample 3
scientific_name
Homo sapiens