title: GSM8745146: CTCF CLIP, UV-crosslinked, NPC, CTCF-ZF1 mutant rescue, rep3; Mus musculus; OTHER
library_construction_protocol: A confluent 15 cm tissue culture plate was washed once with 1x PBS. 10 mL 1x PBS was added to the plate and cells were UV-crosslinked on plate with opened lid, at 400 mJ/cm2 using SpectroLinker XL-1000 (254 nm). Two 15 cm plates were scraped and combined. Cells were pelleted, snap-frozen and stored in -80°C. ±UV cell pellets were resuspended in 20 mL HMSD (20 mM HEPES pH 7.5, 5 mM MgCl2, 25 mM NaCl, 85.5 g/L sucrose, 1 mM DTT) supplemented with protease inhibitors (0.2 mM PMSF, 1 mg/mL Pepstatin A, 1 mg/mL Leupeptin, and 1 mg/mL Aprotinin), and rotated in cold room for 25 minutes. After washing with 1x PBS, nuclei were resuspended in 1 mL cold PBS-lysis buffer (1 mM MgCl2, 0.1 mM CaCl2, 0.5% sodium deoxycholate, 0.5% NP-40 in 1x PBS) supplemented with 1x EDTA-free protease inhibitor cocktail (PIC) (Sigma 11873580001), 40 U/mL Protector RNase inhibitor (Sigma 3335402001), 1 mM DTT, and rotated in cold room for 25 minutes. 20 uL of Turbo DNase (ThermoFisher Scientific AM2238) was added and lysates were incubated at 37°C for 30 minutes, shaking 1100 rpm. Partial RNA digestion was done with a pre-titrated volume of dilute RNase I (ThermoFisher Scientific AM2295), incubated for 3 minutes at 37°C and immediately transferred to ice. 2 uL of SUPERaseIN (ThermoFisher Scientific AM2696) and 0.1% final concentration of SDS was added to stop RNA digestion. Lysates were spun down and supernatant was transferred to new 1.5 mL tubes. Flag-tagged CTCF from supernatant was immunoprecipitated with 5 uL anti-Flag magnetic beads (Millipore Sigma M8823), rotating in cold room for 2 hours. After 4 washes with PBS high-salt wash buffer (1 M NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40 in 1x PBS) and then 4 washes with PBS low-salt wash buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40 in 1x PBS), beads were treated with 0.1 U/uL Turbo DNase supplemented with 1x protease inhibitor cocktail, 0.4 U/uL Roche Protector RNase inhibitor, and 0.1 U/uL SUPERaseIn RNase inhibitor in 1x DNase buffer (ThermoFisher Scientific AM 2239). Beads were incubated at 37oC for 30 minutes, shaking at 850 rpm. For cDNA preparation, oligos used were described previously (10.1016/j.ymeth.2019.10.003). On-beads RNA was dephosphorylated at the 3' end using T4 PNK and ligated to a DNA oligo (L3-App) adapter using T4 RNA Ligase 1 (NEB M0204L). CTCF was eluted from beads twice using 1x flag peptide (5 mg/mL stock) (Sigma F3290) diluted 1:10 in PK buffer (100 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS). Elutions were pooled and proteins were denatured and digested with 4 mg/mL proteinase K incubated in 55oC for 20 minutes, shaking 1200 rpm. 7M final concentration of urea was added and incubated in 55oC for another 20 minutes. RNA-DNA hybrids were extracted using phenol:chloroform:isoamyl alcohol, separating the aqueous phase using phase lock gel tubes (VWR 10847-802). From the aqueous phase, RNA-DNA hybrids were purified by ethanol precipitation overnight at -20oC. RNA-DNA pellet was washed with ethanol and resuspended in nuclease-free water. Reverse transcription (RT) was done using Superscript III Reverse Transcriptase (Invitrogen 18080044). The primer used for RT (RToligo) was complementary to the DNA oligo (L3-App) previously ligated to RNAs. Despite extensive digestion by proteinase K of crosslinked protein-RNA complexes, a small polypeptide remains covalently linked to RNA, which prematurely terminates RT, thereby providing positional information of the crosslinked nucleotides in the resulting cDNA library. Synthesized cDNAs were extracted using Dynabeads MyONE Silane beads (ThermoFisher Scientific 37002D). A second adapter ligation, with barcodes, was done on the 3' end of cDNA using T4 RNA Ligase 1. Adapter-ligated cDNAs were extracted using Dynabeads MyONE Silane beads. The adapter ligations are designed in such a way that sequencing reads start at the position where the cDNA was truncated during reverse transcription. Adapters contained unique molecular identifiers (UMIs) for duplicate removal during analysis. A cDNA pre-amplification was done on beads for 6 cycles using 2x KAPA HiFI Hot Start Mix (KAPA KK2601). The primers used were P5Solexa_s and P3Solexa_s. ProNex® Size-Selective Purification System (Promega NG2001) was used to size select the pre-amplified cDNA and remove primer-dimers. A second PCR amplification was done for 9 cycles using primers P5Solexa and P3Solexa. Libraries were size selected (200-500 bp) with Agencourt AMPure XP beads.