title: GSM8995496: LZ, RAD21L KO, HiC, rep2; Mus musculus; Hi-C
library_name: GSM8995496
library_construction_protocol: Hi-C libraries were generated following standard procedures (1). Briefly, crosslinked populations were incubated in lysis buffer for 30 min in ice and then centrifuged at 2,000 xg for 5 min. Cells were subsequently washed twice with 1xNEBuffer 2 and resuspended in 0.5% SDS in 1xNEBuffer 2. After incubating cells for 10 min at 65ºC, 10% Triton X-100 in 1xNEBuffer 2 was added and incubated for 30 min at 37ºC. Cells were then centrifuged at 2,000 xg for 5 min and washed twice with 1xNEBuffer 2. Following, chromatin was digested by incubating cells overnight with 400U of MboI (NEB) at 37ºC. An aliquot for the ND (Non-Digested) control was taken before the restriction enzyme addition, and another aliquot for the NL (Non-Ligated) control was taken from the digested sample the following day. A digestion control was performed by incubating the ND and NL controls with proteinase K (1 mg/ml) for 1 h at 65ºC, followed by Phenol:Chloroform purification. The digestion quality was checked by running DNA from both controls on a 0.8% agarose gel. After digestion, cells were centrifuged at 2,000 xg for 5 min and washed twice with 1xNEBuffer 2. Chromatin ends were then repaired and biotinylated by resuspending cells with reparation mix (1xNEBuffer 2, 0.05mM dCTP, 0.05mM dTTP, 0.5mM dGTP, 0.05mM biotin-dATP, 50U Klenow) and incubating them for 45 min at 37ºC. Following, cells were incubated for 10 min at 65ºC and centrifuged at 2,000 xg for 5 min. An aliquot for the 3C control was taken before adding the reparation mix. Both the 3C control and the sample were resuspended with ligation buffer (1xNEB T4 ligase buffer, 0.8% Triton X-100, 0.1 mg/ml BSA, 10,000U T4 DNA ligase) and incubated overnight at 16ºC. The following day, cells were centrifuged at 2,000 xg for 5 min and resuspended in 1xNEBuffer 2 with 0.25 mg/ml RNAse A. After incubating cells for 15 min at 37ºC, 0.5 mg/ml proteinase K was added. Cells were then incubated at 65ºC overnight to reverse the crosslink. DNA was subsequently purified with Phenol:Chloroform. DNA quality from the 3C control and the Hi-C sample was assessed by running a 0.8% agarose gel and DNA concentration was measured by a Qubit. For library preparation, samples were first sonicated 30 s ON, 90 s OFF, 4 cycles, low mode (3 times). Subsequently, biotinylated fragments were pulled down using Dynabeads MyOne Streptavidin T1 beads. Briefly, beads were washed with 2xbinding buffer (10mM Tris-HCl pH 7.5, 1mM EDTA, 2M NaCl) and 0.05% Tween 20 in 1xbinding buffer. Beads were then resuspended with 1xbinding buffer, mixed with the sonicated samples and incubated for 30 min at RT under rotation. After two washes with 1xbinding buffer, samples were resuspended with the end repair mix (1xNEB T4 DNA ligase buffer with 10mM ATP, 0.5mM dNTP mix, 50U NEB T4 PNK, 12U T4 DNA polymerase I, 5U Klenow) and incubated for 30 min at RT to repair ends and remove biotin from unligated ends. Beads were then washed twice with 1xbinding buffer, resuspended with the dATP attachment mix (1xNEBuffer 2, 0.5mM dATP, 2.5U NEB Klenow exo minus) and incubated at 37ºC for 30 min. Subsequently, beads were washed twice with 1xbinding buffer, resuspended in 1xNEB Quick ligation reaction buffer together with the NEBNext adapter and Illumina adapters and incubated 15 min at RT to ligate adapters to chromatin ends. Following, beads were washed twice with 1xbinding buffer, resuspended with Tris buffer and mixed with 1 µM primer P5, 1 µM primer P7 and cocktail master mix (NEB). A final amplification was performed following the PCR program: 98ºC 30 s; 98ºC 10 s, 60ºC 30 s, 72ºC 30 s x 8 cycles; 72ºC 5 min. The beads were then captured on a magnet and the supernatant with the amplification products were transferred to a new tube. A 1:1 amount of AmpureBeads was added to the samples and incubated for 10 min at RT. The beads were then washed twice with 70% ethanol, eluded with Tris buffer and incubated for 5 min at RT. AmpureBeads were finally separated on a magnet and the solution was transferred to a new tube. The quality and concentration of the libraries was checked by Qubit and the Agilent Bioanalyzer System. Libraries were submitted for Illumina sequencing (paired-end 150bp each side on NovaSeq 6000).