GSM9049513: CTCF_CT_HIP_935; Homo sapiens; OTHER
All buffers were prepared as described in Kaya-Okur et al., 2019 (PMID: 31036827) without the addition of digitonin. Human hippocampus tissue (30mg) was dounced in 1mL NE1 Buffer with a loose-fitting pestle (5-10 strokes) followed by a tight-fitting pestle (15-25 strokes). Lysates were filtered through 30µM CellTrics filters (Sysmex, Cat#04-0042-2316). After centrifugation for 4 minutes at 1300g at 4°C, nuclei were resuspended in PBS supplemented with Roche c0mplete EDTA-free protease inhibitor (Roche, Cat# 11873580001). 300K nuclei were crosslinked with formaldehyde (Thermo Scientific, Cat#PI28906) at a final concentration of 0.1% at room temperature for 2 minutes, followed by quenching with 1M glycine. Crosslinked nuclei were centrifuged for 4 minutes at 1300g at 4°C and resuspended in 100-300uL of Wash Buffer. 10uL of ConA beads per sample (Bangs Labs, Cat# BP531) were washed 3x in 1mL Binding Buffer and resuspended in Binding Buffer at the original slurry volume. 10uL of beads were added to the nuclei and tubes were rotated for 5-10 minutes at room temperature before placing on a magnetic stand.  Supernatant was removed and beads were resuspended in 100uL Antibody Buffer. CTCF antibody (CST, Cat#3418S) was added at a 1:100 dilution and samples were rotated overnight at 4°C. The next morning, the supernatant was removed and the nuclei were resuspended in 300uL  Was Buffer with secondary antibody (Novus, Cat#NBP1-72763) at a 1:100 dilution. Samples were rotated for 1 hour at room temperature, then washed 3x in 1mL Wash Buffer. After washing, pA-Tn5 (EpiCypher, Cat#15-1017) was added at a 1:50 dilution in 300uL of Dig-300 Buffer and samples were rotated for 1 hour at room temperature followed by washing 3x in Dig-300 Buffer. After removing the supernatant, 300uL Tagmentation Buffer was added to the nuclei and samples were incubated at 37°C for 1 hour with shaking at 500rpm. Tagmentation was stopped by adding 10 uL 0.5 M EDTA, 3 uL 10% SDS, and 2.5 uL 20 mg/mL Proteinase K. Samples were vortexed and incubated for 1 hour at 55°C with shaking at 500rpm to digest and reverse crosslinks. DNA was extracted using a standard Phenol-Chloroform extraction protocol. PCR  was carried out using Nextera i5 and i7 primers and NEBNext HiFi 2x PCR MM (NEB, Cat#M0541S). PCR products were purified using VAHTS DNA Clean Beads (Vazyme, Cat#N411-01) at 1.3x volume and eluted in 10mM Tris-HCl pH 8. Libraries were sequenced on Element AVITI using 2x150 Sequencing Kit Cloudbreak FS Medium Output (860-00012).