title: GSM7504025: HeLa, control ProteinG IP, replicate 2; Homo sapiens; OTHER
library_construction_protocol: DRIPc-seq procedure was based on previously published protocols (PMID: 32544226, PMID: 31053798) with some modifications. 1.25 x 106 HeLa cells were seeded onto 60 mm plates. After 24 hours, the culture medium was removed, and cells were washed once with cold PBS and twice with TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Cell lysis was performed directly on the plate by adding 0.5 mL of lysis buffer (TE supplemented with 0.625 % SDS and 0.0625 mg/mL Proteinase K), and the suspension was immediately transferred to a fresh 2 mL tube and incubated for 4.5 hours at 37°C with gentle mixing every 15 minutes. Afterward, genomic DNA was purified by phenol: chloroform: isoamyl alcohol (25:24:1) extraction and ethanol precipitation. Precipitated DNA was washed with 70% ethanol, air-dried, and resuspended in 30 uL of TE buffer. Next, pure gDNA was sheared by sonication using Bioruptor Plus with a water cooling system (Diagenode) as follows: 3 µg of gDNA diluted to 200 µL in TE was placed in 1.5 mL DNA LoBind Tubes (Eppendorf) and subjected to 30 cycles of sonication 30 s ON, 90 s OFF at a "Low" mode. The length of fragments after sonication was evaluated by running DNA samples before and after shearing on a 1.5 % agarose gel in 1X TAE (40mM Tris pH 8.0, 20 mM acetic acid, 2 mM EDTA) at 70-80 V. Afterward, 4 µg of fragmented gDNA was mixed with 10 µg of  enDR3-3xFLAG or S9.6 antibody, diluted to 1 mL final volume with binding buffer (20 mM Hepes pH 7.4, 150 mM NaCl, 0.05 % Triton X-100), and incubated overnight with gentle rotation in a refrigeration cabinet. The next day, input fractions containing  enDR3-3xFLAG or S9.6 were added to 50 µL of M2 anti FLAG Magnetic Beads (Sigma) or Protein G Dynabeads (Thermo), respectively, and incubated for 2 hours. Additionally, negative controls were prepared by incubating 4 µg of sonicated gDNA with beads without the bait. Following the incubation, the unbound fraction was removed, and beads were washed three times with 500 µL of binding buffer for 5 min with gentle rotation in a refrigeration cabinet. Elution was performed by adding 125 µL of the elution buffer (50 mM Tris pH 8, 1mM EDTA, 0.5% SDS, 0.64 mg/mL Proteinase K) for 45 min at 55°C. Two replicates of each sample were pooled and purified using a DNA clean and concentrator kit (Zymo Research) according to the manufacturer's protocol. After elution with 30 µL of the elution buffer (Zymo Research), half of the eluate was treated with 2 µL of DNAse I (1 U/µL) in 1X DNase I digestion buffer (Thermo Fisher Scientific) for one hour at 37°C. For DNaseI inactivation, 1 µl of 0.5 M EDTA was added for 10 min at 65°C. RNA was purified using the RNA clean and concentrator kit (Zymo Research). ChIP-seq: HeLa Flp-In and derivative cell lines stably expressing enDR3-mEGFP and nb-enDR3-mEGFP were seeded on 100 mm dishes and treated with tetracycline at 25 ng/mL or 38 ng/mL, respectively. After 24 hours, cells were detached with trypsin and counted using hemocytometer. Then, 1.2 x 107 cells were pelleted by centrifugation for 4 min at 300 x g. The culture medium was removed, and cells were washed twice with 5 mL of PBS. Following 4 min centrifugation at 300 x g, cell pellets were suspended in 0.8 mL of cell lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.1% IGEPAL CA-630) supplemented with protease inhibitors cocktail, 1 mM PMSF, and 40 U/mL RiboLock (RL) RNase Inhibitor (Thermo Fisher Scientific), and incubated on ice for 15 min with gentle inverting every 3 min. Then samples were centrifuged at 700 x g for 5min at 4°C. Supernatants were removed and cell nuclei were lysed with 1.1 mL of nuclei lysis solution (20 mM Hepes pH 7.4, 1 mM EDTA, 100 mM NaCl, 5 % glycerol, 0.5 % Triton X-100, 0.1% SDS) supplemented with protease inhibitors cocktail, 1 mM PMSF, and40 U/mL RL. Nuclei lysates were transferred into milliTUBE AFA Fiber (Covaris) and sonicated using S220 Focused-ultrasonicator (Covaris) for 8 min at a Peak Power of 150 Watt, 10% Duty Factor and 200 cycles per burst. Chromatin shearing efficiency was examined by running 800 ng of DNA per well on 1.5 % agarose gel in 1X TAE at 70-80 V. Sonicated samples were transferred from milliTubes to 1.5 mL DNA LoBind tubes (Eppendorf) and centrifuged for 10 min at 20 000 x g at 4°C. Supernatants (hereafter inputs) were transferred to fresh LoBind tubes, and protein concentration was estimated by the Bradford method. For NGS and qPCR analyses, 65 µL (7.5 %) of the input fraction was collected, and 40 µL (4.5%) for the western blot analysis. For immunoprecipitation, 1 mg of input was added to 60 µL GFP-trap magnetic beads (homemade) and incubated with gentle rotation overnight at 4°C. The beads were captured using a magnetic stand, washed three times with 500 µL of low salt washing buffer I (20 mM Tris pH 8, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS) supplemented with 1X protease inhibitors cocktail. Next, beads were washed three times with 500uL of high salt washing buffer II (20 mM Tris pH 8, 2 mM EDTA, 500 mM NaCl, 0.5% Triton X-100, 0.1% SDS) with 1X protease inhibitors cocktail, and once with 500uL of washing buffer III (10 mM Tris pH 8, 1 mM EDTA, 250 mM LiCl, 1% IGEPAL CA-630, 1% Sodium deoxycholate) without inhibitors. Afterward, beads were washed twice with 500 µL of TE buffer (10 mM Tris pH 8, 1 mM EDTA), and 85 µl of the elution buffer (50 mM Tris pH 8, 50 mM NaHCO3, 1% SDS) was added and incubated for 15 min at 65°C with shaking (the 30s ON, 700 rpm/ 2 min OFF). Eluate (E1) was collected and transferred into a fresh 1.5 mL DNA LoBind tube. The elution step was repeated by adding 85 µl of fresh elution buffer, and the second eluate (E2) was pooled with E1. 20 µL of IPed fraction was collected for western blot analysis. Simultaneously, 85 µL elution buffer was added to the 65 µL of the input fractions and incubated for 15 min at 65°C with shaking (30s ON, 700rpm/2 min OFF). Next, 300 mM sodium chloride and 1 µl of RNase A (10 mg/mL) were added to each input and IPed sample and incubated for 45 min at 37°C in ThermoMixer (the 30s ON, 700 rpm/2 min OFF). Then the Proteinase K at a final concentration of 0.4 mg/ml was added, and the reaction was incubated at 55°C for one hour in ThermoMixer (the 30s ON, 1200 rpm/2 min OFF). DNA was purified using Zymo ChIP DNA clean & concentrator (Zymo Research) according to the manufacturer's protocol. The quality of the ChIP procedure was verified by qPCR. For DRIPc-seq, purified RNA was used for stranded NGS library preparation using KAPA RNA HyperPrep Kit (Kapa Biosystems) according to the manufacturer's protocol. For ChIP-seq, purified DNA was used directly for NGS library preparation using KAPA RNA HyperPrep Kit (Kapa Biosystems) according to the manufacturer's protocol starting with the second DNA strand synthesis. DRIPc-seq, ChIP-seq