GSM8669275: MONO, CSF1R Capture-HiC, rep1; Homo sapiens; OTHER
In situ Hi-C in monocytes and HL60 cells was performed using the Arima HiC kit (Arima Genomics), following the user guide for mammalian cell lines. For the crosslinking step, according to the standard input protocol, 5 million cells were crosslinked then flash-frozen in aliquots of 1 million cells. After estimating the DNA input from crosslinked cells, one aliquot of cells (~2 ug of DNA) per condition was sufficient to proceed with the Hi-C protocol. Digestion and proximity ligation steps were done according to the kit protocol. Proximally ligated DNA underwent quality control checks for ligation, sonicated with the Covaris ME220 sonicator, then purified and size selected with AMPure XP Beads (Beckman Coulter) to obtain a fragment size distribution of 200-600bp prior to the biotin enrichment step. For library preparation, end repair, dA-tailing, and adaptor ligation were performed using the NEB Next Ultra II DNA Library Prep kit for Illumina, following a protocol tailored for Arima Hi-C DNA. Capture Hi-C of the CSF1R gene locus was performed on Hi-C libraries from either the primary cells or HL-60 CRISPR clones using the myBaits Hybridization Capture for Targeted NGS kit (Arbor Biosciences).  Biotinylated RNA baits were designed to target a 35,413 bp sequence which included the genomic locus of the NM_001288705.2 transcript of CSF1R plus 1000bp upstream and downstream of the transcript. A total of 1,474 70-nucleotide (nt) baits were designed with 3X tiling. Raw sequences were BLASTed against the human genome to filter out 123 baits that did not meet stringent analysis criteria. To amplify and remove residual biotinylated nucleotides from the library samples for myBaits compatibility, Hi-C libraries were first PCR amplified for 7 cycles with the following conditions: 98°C for 2 min, 7 cycles of 98°C for 20 sec, 60°C for 30 sec, and 72°C for 60 sec, then a final extension of 72°C for 5 min. The reaction included 200ng of library DNA, 2X KAPA HiFI HotStart ReadyMix (Roche), and P5 and P7 universal primers (500nM). Amplification reactions were then incubated with prewashed Dynabeads MyOne Streptavidin C1 beads (Invitrogen) for 15 minutes, followed by collection of the supernatant for purification with Ampure XP beads. To the supernatant, 1.8X reaction volume of beads were added then incubated for 5 mins at room temperature. Samples were then placed on a magnetic separator for 10 mins then washed twice with 70% ethanol. Following the washes, beads were air-dried for 20 mins then DNA was eluted in 40uL of 0.1X TE. CSF1R capture of the purified libraries was then performed with the myBaits kit with some modifications to the manufacturer's protocol. The hybridization step was done at 62°C for 25 hours. The final purification step was done using AMPure XP beads, following the same procedure done after pre-amplification step.