GSM3665791: 4C-seq_Enhancer1_CRISPR-clone_Rep1; Mus musculus; OTHER
Briefly, 10 million cells were cross-linked for 10 minutes with 2% paraformaldehyde in ES-culture medium, quenched with glycine and lysed in 30 ml lysis buffer (10mM Tris pH 7.5, 10mM NaCl, 0.2% NP-40, 1X protease inhibitors) for 30 minutes at 4°C  . Nuclei were then incubated with 0.25% SDS for 30 minutes in NEB buffer3 followed by Triton X-100 treatment for 30 minutes both at 37°C. Nuclei were then digested with 700 units DpnII enzyme (NEB) overnight at 37 ̊C. Enzyme was inactivated at 65 ̊C for 15 min followed by  'in nuclei' ligation at 16 ̊C with 2,000 units T4 ligase (NEB)66. Samples were then treated with protease K and RNAseA and DNA was purified by phenol–chloroform (Sigma: 77618-500ml). DNA was further digested with 50 units BfaI, purified with QIAquick PCR purification columns followed by second ligation at 16°C. Next, 1000 ng of 4C-seq library was amplified with bait-specific inverse primers using Expand Long template PCR sytem (Roche 11759060001) for 28 PCR cycles. PCR products were then purified and 50 ng DNA was used for library preparation using KAPA Hyperprep kit (Roche 07962363001) and 5 PCR cycles.  Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C.Subsequently adapters were  ligated  by adding 30µl ligation buffer, 10 Kapa l DNA  ligase, 5µl diluted adaptor  in a total volume of 110µl and incubated for 15 minutes at 15°C.Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and were size selected using bead purification protocol (200bp-1500bp).  Correct size selection was confirmed by BioAnalyzer analysis.   Sequencing was performed using Illumina Nextseq500  machines and generated 50bp pair end reads.
GEO
GSM3665791