title: GSM4848655: CRISPR_Con_LPS2h_Ccl2_e2_bait_rp1; Mus musculus; OTHER
library_construction_protocol: WT or LNA control cells, CRISPR KO cells , Ccl2 E1 eRNA knockdown cells and GPS2/SMRT knockdown cells  were re-suspended with 10 ml lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100, for 10 minutes. Nuclei were re-suspended into 440 l Milli-Q water and 60 l of digestion buffer (15 l 10% SDS and 75 l 20% Triton X-100) was added. The cell lysis mixture was then incubated at 37 C for 2 hours in the shaker (900 RPM). 200U DpnII (NEB, R0543M) was added into the mixture three times. The lysis mixture was then incubated in the shaker overnight. The reaction mix was inactivated after overnight digestion by incubating at 65C for 20 min. The samples were then transferred to 50 ml Falcon tubes with 700 l ligation buffer and 5.5 ml Milli-Q water. The samples were then supplemented with 100 U T4 ligase (NEB, M0202M) and incubated overnight at room temperature.  After that, samples were de-crosslinked at 65C for 12 hours. The ligated DNA was then purified by phenol-chloroform method, and further digested with NlaIII (NEB, R0125L) at 37 C overnight. The restriction enzyme was then inactivated for 20 min at 65 C. The DNA was further ligated at room temperature overnight. After the second ligation, the DNA was purified by QIAquick PCR purification kit PCR purified DNA (by baits) was processed using Takara SMRTer ThruPLEX DNA-seq Kit. Briefly, DNA was end-repaired and ligated to stem-loop adaptors. Then DNA was PCR amplified with Illumina-compatible primers for 7-11 cycles and library fragments of 200 to 400 bp were selected using Ampure XP beads.
attributes.tag: GEO Accession
attributes.value: GSM4848655