title: GSM4715063: Unstim_CF_Peak9_ViewPoint_4C; Mus musculus; OTHER
library_construction_protocol: After 48h of TGFb treatment, fibroblast cells were washed 3 times with cold PBS and then sequentially cross-linked with 1% formaldehyde for 10 min. The reaction was quenched by addition of glycine to a final concentration of 0.125 M. Cells were washed once again with cold PBS and nuclei were extracted by incubation 10min in ice with 5 ml of ice-cold lysis buffer (10 mM Tris–HCl, pH 8.0, 10 mM NaCl, 0.2% NP-40 + protease inhibitor). Nuclei were resuspended in restriction enzyme buffer (NEB Cat#B7006S) and incubated with 0.3 % SDS for 1h at 37 C and further incubated with 2% Triton X-100 for 1h. 400U of DpnII restriction enzyme (NEB Cat#R0543S) was added and incubated overnight. Restriction enzyme was heat inactivated at 65C for 20 min. Ligation of DNA regions in close physical proximity was performed using 1000U of T4 DNA ligase (NEB M0202M) overnight. The following day, 300µg of proteinase K were added and decrosslinking was performed at 65C overnight. After de-crosslinking, DNA was purified using phenol/chloroform precipitation and the second digestion was performed by adding 400U of NlaIII restriction enzyme (NEB Cat#R0125S), then incubation at 65C for 4h. After the second digestion, ligation of DNA was performed again using T4 DNA ligase as described above. 4C-seq libraries were amplified using PCR with primer containing partial Illumina sequence adaptors (1st primer: gttcagagttctacagtccgacgatc; 2nd primer: agacgtgtgctcttccgatct). The first primer was designed on each view point and the second primer designed beside the closest NlaIII cutting site to the view point. Full-length Illumina sequencing adaptors and barcodes were added by the second round of PCR. Finally, libraries were quantified by Qubit and library quality checked with bioanalyzer Agilent 2100 (Agilent Technologies). High-throughput sequencing was done using a SE75 run on a NextSeq 500 instrument (Illumina
attributes.tag: GEO Accession
attributes.value: GSM4715063