GSM9337285: CHiC - CN BxC rep4; Mus musculus; Hi-C
Adherent cells were washed with PBS two times and scraped into 5 mL PBS. / Dissected mouse cortices (two BxC and two CxB) were snap frozen in liquid nitrogen and stored at -80 °C. Frozen tissues were pulverized using Cell crusher in liquid nitrogen. Samples resuspended in 5 mL PBS and crosslinked in 2% formaldehyde by adding 500 μL of TC buffer and incubating at room temperature for 20 min. Reaction was quenched by adding 289 μL of strop solution I and incubating at RT for 5 min. Crosslinked tissue powder was then centrifuged at 2,000 xg at RT for 15 min and supernatant was discarded. Pellet was stored at -80 C until use. Samples were resuspended in 20 μL lysis buffer, lysed at 4 C for 30 min, conditioned using SDS solution at 62 C for 10 min, and quenched by incubating with 20 μL of stop solution II at 37 C for 15 min. Digestion of extracted chromatin was performed using Arima enzymes by incubating at 37 C 60 min, 65 C 20 min, and 25 C 10 min. Digested chromatin was subject to biotin end repair at RT for 45 min, followed by proximity ligation at RT for 15 min. Crosslinking reversal was performed using proteinase K and incubating at 55 C 30 min, 68 C overnight, and 25 C 10 min. Proximity-ligated DNA was purified using AMPure XP beads. Proximity-ligated DNA was brought to 100 μL in elution buffer and fragmented using Covaris S220 (140 W, 10% duty factor, 200 cycles per burst, 55 sec) to obtain a target fragment of 400 bp. Double sided size selection was performed using 0.6X and 0.25X AMPure XP beads. Biotin enrichment was performed using 300-400 ng of size-selected DNA and 12.5 μL T1 Streptavidin beads in 200 μL binding buffer reaction. Two washes were performed in 200 μL wash buffer at 55 C for 2 min each. Final wash was performed in 100 μL of elution buffer. Washed beads were resuspended in 50 μL of water. On beads end repair was performed by incubating end repair mix at 20 C 15 min 72 C 15 min, followed by adaptor ligation at 20 C 30 min. Samples were cleaned up by magnetizing beads and washing in 200 μL wash buffer at 55 C 2 min and in 100 μL elution buffer. Washed beads were resuspended in 34 μL of water and immediately used for library amplification using Herculase II Fusion polymerase (98 C 2 min / 10X 98 C 30 sec 60 C 30 sec 72 C 1 min / 72 C 5 min). Different index primer combinations were used for each sample. Amplified library was purified using 1X AMPure XP beads. An aliquot of 1.5 μg PCR product in 30 μL elution buffer was pre-cleared of biotin using 50 μL T1 Streptavidin beads in 230 μL reaction. Pre-cleared Hi-C library was resuspended in 14 μL water. Approximately 1 μg of Hi-C library was incubated with custom probes (Agilent Technologies) in thermocycler (95 C 5 min, 65 C 10 min / 60X 65 C 1 min 37 C 3 sec) and purified using 50 μL T1 Streptavidin beads. Vigorous washes were performed six times at 70 C 5 min each. Capture enriched library is amplified using Herculase II polymerase (98 C 2 min / 13X 98 C 30 sec 60 C 30 sec 72 C 1 min / 72 C 5min). Final library purification was performed using 1X AMPure XP beads and resuspended in 25 μL water. Samples were pooled equimolar. Sequencing was performed PE150.