title: GSM8251645: DRIPseq, CFAP20KO, mock, rep2; Homo sapiens; OTHER
library_construction_protocol: After trypsinization, pellets were lysed in TE buffer 0.2% SDS supplemented with 0.04 mg/mL Proteinase K (life technologies, EO0491) at 37°C overnight. Samples were centrifuged and DNA was extracted from the lysates with phenol chloroform isoamyl alcohol (25:24:1, Thermo Fisher, 10103971). The extracted DNA was then precipitated with 1/10 volume 3M Sodium Acetate pH 5.2 and 2.5 volume ethanol. White DNA particles were rescued with a cut 1 mL tip and washed 3 times with 80% ethanol. Dried DNA was restituted by incubation in TE buffer at 4° overnight. For enzyme digestion, samples were incubated with HindIII-HF, EcoRI-HF, BsrGI-HF, SspI-HF and XbaI (NEB, R3104, R3101, R3575, R3132, R0145 respectively) 30 U each in NEB buffer 2.1 with 0.2 mg/mL BSA (NEB, B9000) overnight at 37°C. Digested DNA was then extracted by adding 0.4 volume water and 1 volume phenol chloroform isoamyl alcohol (25:24:1). The aqueous phase after extraction was incubated with 33 μg/mL glycogen (Thermo Scientific, R0561), 1/10 volume 3M Sodium Acetate pH 5.2 and 2.5 volumes ethanol overnight at -20°C to precipitate the DNA. Samples were centrifuged at 16000 g for 35 min at 4ºC and the supernatant was discarded. After air drying, pellets were resuspended in TE buffer for 1 h on ice. For RNaseH1 control, 10 μg of sample was collected in pre-lubricated tubes and treated with 3 μl RNaseH1 (NEB, M0297) and 6 μl RNaseH1 reaction buffer (NEB, B0297) in a total volume of 50 μl in TE buffer overnight at 37°C. 8 μg DNA was dissolved in a total volume of 500 μl TE buffer and from this, 50 μl input sample was collected. For the R-loop immunoprecipitation 10 μl DNA-RNA Hybrid [S9.6] Antibody was added to 8 μg DNA in pre-lubricated tubes in a total volume of 50 μl in 10x binding buffer (100 mM sodium phosphate pH 7, 1.4 M sodium chloride, 0.5% Triton X-100) and incubated overnight at 4°C in a rotating shaker (11rpm). Per sample, 100 μl Dynabeads™ Protein A (Themo Fisher, 10001D) were washed twice with 1x binding buffer (10x binding buffer diluted in TE buffer) for 10 min at RT. The beads were then added to the DNA and incubated at 4°C in a rotating shaker (11rpm) for 2 h. By using a rotator at RT, the beads were washed twice in 1x binding buffer for 15 min. To elute the DNA, the beads were incubated in 300 μl DRIP elution buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 0.5% SDS) supplemented with 7 μl Proteinase K at 56°C on a rotator for 45 min. The eluted DNA was then first purified with 1 volume phenol chloroform isoamyl alcohol (25:24:1) followed by a cleanup with 33 μg/mL glycogen, 1/10 volume 3M sodium acetate pH 5.2 and 2.5 volumes ethanol. The aqueous phase was then incubated overnight at -20°C to precipitate the DNA. Samples were centrifuged at 16000 g for 35 min at 4°C and the precipitates were cleaned with 80% ethanol at RT. After air drying, pellets were gently resuspended in 200 μl TE buffer and left on ice for a few hours. The samples were sonicated by using the Bioruptor Pico (Diagenode), 6 cycles of 15s ON/60 s OFF in a 4°C water bath. The final sample was purified using a Qiagen MinElute kit (Quiagen, 28004) and concentrations were measured on the Qubit 2.0 (Invitrogen) with the use of the Qubit 1x dsDNA HS assay kit (Thermo Fisher, Q33230). Sample libraries were prepared using Hifi Kapa sample prep kit and A-T mediated ligation of Nextflex adapters or xGen UDI-UMI adapters. Samples were sequenced using an Illumina NextSeq500 or HiSeq X, using paired-end sequencing with 42 or 151 bp from each end.