title: GSM5491589: HiC_iXEN_48hr; Mus musculus; Hi-C
library_construction_protocol: 1 million cells per sample were trypsinized, washed in growth media and fixed for 10 minutes at room temperature while rotating with 1% formaldehyde (Thermo: 28908) in 1ml of HBSS media. To stop fixation, Glycine was added at final concentration of 0.13M and incubated for 5 minutes at RT and 15 minutes on ice. Cells were then washed once in cold PBS, centrifuged at 2500g 4oC for 5 mins (these centrifugation conditions were used for all washes following fixation) and pellets frozen at -80oC. Thawed cell pellets were incubated in 1ml lysis buffer (10mM Tris-HCL pH8, 10mM NaCl, 0.2% Igepal CA-630, Roche Complete EDTA-free Sigma #11836170001). Following lysis, cells were dounced for a total of 40 strokes with the “tight pestle” and then washed in cold PBS. For DpnII digest, cells were resuspended in 50µl 0.5% SDS and incubated at 62oC for 10 minutes. Then 150µl of 1.5% Triton-X was added and cells incubated for 15 minutes at 37oC while shaking at 900rpm. 25µl of 10X DpnII restriction buffer (NEB) was added, and cells further incubated for 15 minutes while shaling. 200U of DpnII (NEB R0543M) were then added and incubated for 2hours, then 200U more and incubated overnight. Next morning 200U more were added and incubated for 3h (total 600U of DpnII). DpnII was inactivated at 62oC for 20 minutes. Biotin fill-in was done by incubating cells with a mixture of 4.5 µl dCTP dTTP and dGTP at 3.3 mM, 8µl klenow polymerase (NEB M0210L) and 37.5µl Biotin-14-dATP (Thermo 19524016) for 4h at RT while shaking at 900rpm for 10 seconds every 5 minutes. Ligation was done overnight at 16oC also rotating at 900rpm for 10 seconds every 5 minutes by adding 120µl of 10X ligation buffer (NEB), 664µl water, 100µl 10% Triton-X, 6µl BSA 20mg/ml, and 2µl T4 ligase (NEB cat #M0202M). For Capture-C, biotin fill-in step was skipped and 50µl more of water was added to the ligation mix. Crosslink removal was done overnight with 50µl of proteinase K in 300µl of following buffer (10mM Tris-HCl pH8.0, 0.5M NaCl, 1%SDS) while shaking at 1400rpm at 65oC. Following Sodium Acetate and 100% Ethanol -80oC precipitation, DNA was resuspended in 50µl 10mM Tris HlL for Hi-C or 130µl for Capture-C. Sonication for Hi-C was done using Covaris onetube-10 AFA strips using the following parameters for a 300bp fragment size (Duration: 10secs, repeat for 12 times, total time 120 secs, peak power-20W, duty factor 40%, CPB-50). Sonication for Capture-C was done using Covaris AFA microtubes 130 with following settings for a fragment size of 200bp fragments (Duration: 225Secs, peak power-75W, duty factor 25%, Cycles per Burst-1000). Sonications were performed in a Covaris ME220 sonicator. Sonicated material was then size selected using SPRI beads with the following ratios: 0.55X and 1X for Capture-C and 0.55X and 0.75X for Hi-C. Hi-C material was then bound, washed and recovered to 150µl Streptavidin C1 beads (Thermo 65002) per sample following manufacturer's recommendations. Bead-bound DNA was resuspended in 50µl 10mM Tris HCl. Library preparation was done using Kapa Hyper Prep KK8502 kit. 10µl of End-repair buffer and enzyme mix were added to resuspended beads and incubated for 30 minutes at RT and then 30 minutes at 65oC. 1µl of 15mM annealed-Illumina adaptors, containing an universal p5 and an indexed p7 oligo, were then incubated with a mixture containing 40µl of ligase and ligation buffer at RT for 60 minutes. Libraries were then amplified using 4 reactions per sample for a total of 200µl and 10 cycles, as recommended by manufacturer. For Capture-C, following sonication and size selection, 1µg of template material was resuspended in 50µl of 10mM Tris and used for library prep with 10µl of End-Repair reaction. 5µl of 15mM annealed -Illumina adaptors were ligated to the Capture-C material. Using a total volume of 100µl, library was amplified by PCR using 6 cycles. For capture, 1µg of Capture-C library per sample was mixed with mouse COT1 DNA and universal as well as index-specific blocking oligos from SeqCap EZ HE-oligo (Roche). 4.5µl pool of biotinylated probes (xGen Lockdown Probe Pools from IDT), with each probe at 0.4fmol/µl targeting the promoters of our loci of interested were added to this mixture and incubated for 3 days at 47oC. Following binding to Streptavidin C1 beads, material was washed as recommended by the SeqCap EZ Hybridization and Wash Kits. Following washes material was amplified by PCR using Kapa polymerase and 14 cycles. Material from different samples was then combined and 1µg of pooled libraries was recaptured in a single reaction and amplified with 8 cycles. Paired-end 50.