GSM5268046: Satb1_cKO_rep2_HiC; Mus musculus; Hi-C
Both Hi-C and HiChIP experiments were performed identically until the chromatin immunoprecipitation step. Aliquots of 10 million isolated thymocytes resuspended in 1X PBS were fixed by adding 1/10th sample volume of fixation butter [11% methanol-free formaldehyde (Pierce, 28908), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes pH 8.0] and rocking at RT for 10 minutes. To quench the reaction, glycine was added to 0.125 M final concentration and incubated at RT for 5 minutes, while rocking. After two washes with 1X PBS, cell pellet was resuspended in 500 μl of ice-cold Hi-C Lysis Buffer (10 mM Tris-HCl – pH 8, 10 mM NaCl, 0.2% NP40, 0.5 mM PMSF) and rotated at 4°C for 1.5 hours. Cells were centrifuged at 2,500 g, at 4°C for 5 minutes and the supernatant was discarded. The cell pellet was washed once with 500 μl of ice-cold Hi-C Lysis Buffer and then resuspended in 100 μl of 0.5% SDS. Cells were incubated at 62 °C for 10 minutes and then combined with 296 μl of H2O and 50 μl of 20% Triton X-100. Samples were incubated at 37°C for 15 minutes and then combined with 50 μl of 10X DpnII Buffer and 200 U of DpnII restriction enzyme (NEB, R0543M) and digested for additional 16 hours at 37 °C while shaking (160 rpm). The restriction enzyme was inactivated at 62°C for 20 minutes and the nuclei were centrifuged at 2,500 g, at 4 °C for 6 minutes. The supernatant was discarded and the nuclei were resuspended in 300 μl Fill-in Buffer [30 μl Klenow Buffer 10X (NEB, M0210L), 238.5 μl water, 15 μl 1 mM Biotin-16-dCTP (Jena Bioscience, NU-809-BIO16-L), 1.5 μl of each 10 mM dATP, dGTP, dTTP (Promega, U1240), 12 μl 5 U/μl DNA Polymerase I, Klenow Fragment (NEB, M0210L)].  The biotinylation mixture was incubated at 37°C for 30 minutes with rotation. SDS was added to a final concentration of 0.5% to inactivate the Klenow enzyme. Triton X-100 was added to 1% final concentration and samples were incubated at 37°C for 5 minutes. Samples were centrifuged at 2,500 g, at 4 °C for 10 minutes and the supernatant was discarded. The nuclei pellet was resuspended in the Ligation Buffer [120 μl 10X NEB T4 DNA Ligase Buffer with 10 mM ATP (NEB, B0202), 60 μl 20% Triton X-100 (1% final), 6 μl 2% (20 mg/ml) BSA, 40 μl 30% PEG 6,000 (1% final), 5 μl 400 U/μl T4 DNA Ligase (NEB, M0202L), 969 μl water]. Samples were incubated for 6 hours at RT with rotation. Nuclei were centrifuged at 2,400 g, at RT for 15 minutes and the supernatant was discarded. The pellet was resuspended in 60 μl Lysis Buffer (1% SDS, 50 mM Tris-HCl – pH 8, 20 mM EDTA, 1X protease inhibitors) and incubated at RT for 15 minutes. The lysate was diluted to 600 μl using ice cold TE Buffer supplemented with protease inhibitors and then sonicated with a Labsonic M – Tip sonicator for 3 cycles (30 seconds ON/OFF, 40% power). The sonicated material was centrifuged at 16,000 g, at RT for 15 minutes and the supernatant was collected into a new tube. Samples from the same genotype were merged and then split again: separately 100 μl for Hi-C and 450 μl for HiChIP.  Hi-C samples were combined with two volumes of Hi-C Elution Buffer (10 mM Tris-HCl – pH 8, 5 mM EDTA, 300 mM NaCl, 1% SDS) and incubated at 65°C overnight. De-crosslinked material was diluted to 500 μl with TE Buffer and treated with RNase A and Proteinase K. DNA was purified using a ChIP DNA Clean & Concentrator kit following the manufacturer's instructions (Zymo Research, D5205). Purified DNA was quantified using Qubit dsDNA BS Assay Kit (Invitrogen, Q32853) checked on an agarose gel for shearing efficiency and 100 μg were used for library construction.  HiChIP samples were combined with Triton X-100 to 1% final concentration and samples were incubated at 37°C for 15 minutes. Samples were combined with an equal volume of 2X ChIP Binding Buffer (20 mM Tris-HCl – pH 8, 2 mM EDTA, 0.2 % sodium deoxycholate, 2X protease inhibitors). Chromatin preclearing and antibody binding to magnetic beads were performed as described in the ChIP protocol. The following antibodies were used for the immunoprecipitation step: SATB1 long isoform (8 μg, custom-made from Davids Biotechnologies), CTCF (7 µg, Abcam, ab70303), H3K27ac (2 µg, Abcam, ab4729). Antibody-coupled beads were incubated with chromatin at 4°C with rotation for 16 hours. Beads were washed five times with ice cold RIPA Buffer (50 mM Hepes pH 8, 1% NP-40, 0.70% Na-Deoxycholate, 0.5 M LiCl, 1 mM EDTA, protease inhibitors) and then twice with TE Buffer. After the first wash with TE Buffer, resuspended beads were transferred into a new tube. Immune complexes bound to beads were eluted in 125 μl Hi-C Elution Buffer (10 mM Tris-HCl – pH 8, 5 mM EDTA, 300 mM NaCl, 1% SDS) at 65°C for 16 hours. De-crosslinked material was diluted to 250 μl with TE Buffer and treated with RNase A and Proteinase K as previously described. DNA was purified using a ChIP DNA Clean & Concentrator kit following the manufacturer's instructions (Zymo Research, D5205). Purified DNA was quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854) and used for library construction. Samples were brought to 25 μl with water. 5 μl and 20 μl for HiChIP / Hi-C samples, respectively, of Dynabeads MyOne Streptavidin C1 beads (Invitrogen, 65001) were washed with 500 μl Tween Wash Buffer (5 mM Tris-HCl – pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20). Beads were resuspended in 25 μl of 2X Biotin Binding Buffer (10 mM Tris-HCl – pH 7.5, 1 mM EDTA, 2M NaCl) and combined with samples. Samples were incubated at RT for 20 minutes. Beads were separated on a magnet and washed twice with 400 μl of Tween Wash Buffer. Beads were washed by 100 μl of 1X TD Buffer (10 mM Tris-HCl – pH 7.5, 5 mM MgCl2, 10% Dimethylformamide). Beads were resuspended in 25 μl of 2X TD Buffer and combined with Tn5 enzyme from the Nextera DNA Sample Preparation Kit (Illumina, FC-121-1030) and water to final volume 50 μl. The amount of Tn5 enzyme was adjusted according to the input DNA amount: 4.5 μl for Hi-C libraries, 1.5 μl for SATB1 HiChIP and 1 μl for other HiChIP libraries. The reaction was incubated at 55°C for 10 minutes. The beads were collected with a magnet and the supernatant was discarded. Beads were resuspended in 300 μl of Strip Buffer (0.15% SDS, 10 mM Tris-HCl – pH 8, 50 mM EDTA) and incubated for 5 minutes at RT to strip off and deactivate Tn5. Beads were washed once with 400 μl of Tween Wash Buffer and once with 500 μl of 10 mM Tris-HCl (pH 8.0).  The beads were resuspended in 50 μl of the following PCR master mix with indexed primers from the Nextera DNA Sample Preparation Index Kit (Illumina, FC-121-1011): 25 μl Phusion HF 2X (NEB, M0531L), 1 μl (1.5 for Hi-C) Nextera Index 1 N7XX, 1 μl (1.5 for Hi-C) Nextera Index 2 N5XX, 23 μl (22 for Hi-C) water. The PCR reaction was performed according to the following program: initial incubation at 72°C for 5 min and 98°C for 1 min, then x cycles of 98°C for 15 s, 63°C for 35 s and 72°C for 60 s. The number of PCR cycles was estimated based on post-ChIP quantification and amplification was 6 cycles for Hi-C libraries, 11 cycles for SATB1 HiChIP and 13 cycles for the other HiChIP libraries. DNA libraries were purified and size-selected using AMPure XP beads, quantified by Qubit and analyzed on a Bioanalyzer. The DNA Libraries were sequenced on an Illumina® HiSeq 4000 2x 75 bp platform.