GSM7856851: SUDHL4_4Cseq_DpnII_CviQI_BCL6p_rep1; Homo sapiens; OTHER
Cells were crosslinked with 2% formaldehyde in PBS at room temperature for 10 minutes, followed by glycine quenching, washing in PBS, isolation of nuclei by hypotonic buffer lysis, and resuspension in primary restriction enzyme buffer. Primary digestion was accomplished by sequential addition of SDS, Triton-X100, and primary restriction enzyme while mixing at 37°C, followed by heat inactivation at 65°C. Ligation was performed after sample dilution in T4 ligase buffer by addition of T4 ligase at 16°C. Ligated DNA was isolated by proteinase K and RNAase A digestion, phenol-chloroform extraction, and ethanol precipitation. DNA was then digested with secondary restriction enzyme, heat inactivated, ligated with T4 ligase, and isolated by ethanol precipitation and QIAquick PCR purification columns. PCR (ExTaq, Takara) with barcoded illumina adaptors was used to amplify sgRNA sequences from genomic DNA corresponding to 500 cell equivalents per library sgRNA, aliquoted into 50 uL PCR reactions (6ug DNA per reaction). All reactions from each replicate were pooled, and 100 ul was gel-purified with E-gel size-select II. To generate each 4C-Seq library, a total of 3.2 micrograms of 4C DNA was amplified across 16 separate 50-microliter PCR reactions containing Q5 high-fidelity polymerase mix (NEB) and oligonucleotides pairs consisting of universal illumina adaptor sequences, sample-specific barcodes, and viewpoint-specific primer sequences. Equal portions of each reaction were pooled and the PCR-amplified library was purified with 0.8 volumes of Ampure XP per volume of PCR product.