GSM7438399
Cells were seeded in 150mm dishes and grown till 70-80% confluency. Media was aspirated and cells were washed with PBS and fixed with 10ml 1% formaldehyde (ThermoFisher 28906) in PBS at room temperature for 5min, with agitation every 2min. The fixing reaction was then quenched by adding 562ul of 2.5M glycine (Thermo J16407.A1) and incubated at room temperature for 5min, with agitation every 2min. The quenched solution was removed and fixed cells were washed twice with 10ml cold PBS. Cells were scraped in 10ml cold PBS+1%BSA and transferred to 50ml tubes pre-blocked with PBS+1%BSA. 5ml cold PBS+1%BSA was used to harvest and residual cells from plates. Cells were pelleted at 300rcf 4degC for 5min and resuspended in 1ml cold PBS+1%BSA. 2e7 cells were counted and transferred to a new cold LoBind tube before being washed twice by resuspending in 1ml cold PBS and centrifuging at 300rcf 4degC for 5min. Cells were resuspended in 0.9ml IP lysis buffer (50mM Tris pH 8, 100mM NaCl, 1.0% IGEPAL, 0.1% SDS, 0.5% sodium deoxycholate, 1mM EDTA 1x Complete protease inhibitor) and lysed on ice for 15min. Lysate was split into 3x300ul aliquots in pre-chilled LoBind tubes and sonicated at high setting for 15min (30s ON 30s OFF) using the Bioruptor Plus (Diagenode B01020001). SOnicated lysate was recombind and clarified via centrifuging at 13krpm 4degC for 10min. Meanwhile, 100ul Dynabead-Protein A (ThermoFisher 10001D) was washed twice in 1ml IP lysis buffer. Beads were resuspended in 200ul IP lysis buffer and split into 2 portions. 1 portion (“pre-clear beads”) was incubated with the clarified lysate at 4degC for 1hr to pre-clear the lysate. To the other portion, 10ug of anti-PCF11 antibody (Abcam ab134391) was added and mixed by tumbling at room temperature for 1hr (“IP beads”). IP beads were washed twice with 500ul IP lysis buffer. Both pre-clear beads and IP beads were magnetized and the pre-cleared lysate supernatant from the pre-clear beads was transferred to the decanted IP beads to be tumbled at 4degC overnight. 20ul of this mixture was set aside as “INPUT” and mixed with 80ul H2O and 100ul 2x elution buffer(). The remainder IP beads was washed once with 1ml low-salt buffer (50mM Tris pH 8, 100mM NaCl, 1% IGEPAL, 0.1% SDS, 0.5% sodium deoxycholate, 1mM EDTA), twice with 1ml mid-salt buffer (50mM Tris pH 8, 250mM NaCl, 1% IGEPAL, 0.1% SDS, 0.5% sodium deoxycholate, 1mM EDTA), twice with 1ml high-salt buffer (50mM Tris pH 8, 500mM NaCl, 1% IGEPAL, 0.1% SDS, 0.5% sodium deoxycholate, 1mM EDTA) and once again with 1ml low-salt buffer with a tube-change. Decanted beads were then eluted with 100ul 1x elution buffer (), shaking at 1krpm 30degC for 15min, before deads were magnetized and the elution supernatant was collected. A second elution was performed and the elutions were combined for a total of 200ul. 8ul 5M NaCl and 4ul 10mg/ml Rnase A (ThermoFisher EN0531) was added to both INPUT and IP elutions before shaking at 1krpm 65degC overnight. 4ul 20mg/ml proteinase K was added and the reactions shaken at 1krpm 60degC for 1hr before allowing them to cool to room temperature. DNA was purified with MinElute PCR cleanup kit (Qiagen 28004) and quantified with Qubit. Illumina sequencing libraries were constructed using KAPA hyper-prep kit (Roche 07962347001), including a dual size selection step according to the manufacturer's protocol. Libraries were quantified using KAPA library quantification (Roche KK4824), pooled and sequenced on Illumina Nextseq PE75.