GSM7419743: DNAseq_WN.rep3; Homo sapiens; OTHER
Approximately 4 million cells were collected from 10 cm culture dishes by rinsing the plates with cold PBS twice and applying 1.5 mL of buffer A (100 mM Tris-HCl pH 7.4, 100 mM NaCl, 30 mM MgCl2, 0.2% Triton, 0.5 mM DTT, 100 mM sucrose) to the plates. The cells were then scraped off and transferred directly to dounce homogenizers, where they were homogenized by applying 40 strokes, 1 second up and 1 second down using the tight pestle (pestle B). After adding 1.5 mL of buffer B (100 mM Tris-HCl pH 7.4, 100 mM NaCl, 30 mM MgCl2, 0.1% Triton, 0.5 mM DTT, 250 mM sucrose) and mixing, the A/B buffer cell pool was layered onto the top of 8 mL of 1 M sucrose cushion in a 15 mL falcon tube, with minimal mixing of the cell suspension with the sucrose cushion. The samples were then centrifuged at 1000 x g for 5 min at 4°C to pellet nuclei, and supernatants were discarded. The nuclei were resuspended in 250 µL of MNase buffer (50 mM Tris-HCl, 5 mM CaCl2 pH 7.9 @ 25°C) supplemented with 100 µg/mL BSA and the resuspended nuclei were re-pooled. A 50 µL mix was taken out and saved as a whole nuclei (WN) fraction. The remaining 200 µL sample was sonicated for 20 total seconds, with 1 second on and 1 second off at 50% power, resulting in 10 seconds of total sonication. A 10 µL mix was taken out and saved as sonicated untreated nuclei for quality control analysis. To the 200 µL sonicated nuclei, 4 µL of the 1.25 U/µL MNase enzyme dilution in 1x MNase buffer was added. The samples were incubated for 5 minutes at 37°C. The samples were immediately placed on ice and 12.5 µL of 100 mM EGTA was added at a final concentration of 6.25 mM to terminate MNase activity. A 10 µL of the mix was saved for the quality control of sonicated treated nuclei. ~200 µL of light MNase-treated fraction was spun at 3000 x g for 3 minutes. Then, 50 µL of the supernatant from the light MNase-treated fraction should be taken out and saved for quality control. All remaining supernatants were removed. The pellet was resuspended in 50 µL of buffer D (50 mM Tris-Cl pH 8.0, 25% (v/v) glycerol, 5 mM MgAc2, 0.1 mM EDTA, 5 mM DTT) for nuclear condensate (NC) fraction. 50 µL of WN fraction from the previous step was also spun at 3000 x g for 3 minutes and resuspended in 50 µL of buffer D. 5 µL of the NC and WN fractions were saved for quality control analysis. Finally, the WN and NC fractions were rapidly frozen in liquid nitrogen and stored at -80°C. For DNA-seq, Genomic DNA from the whole nuclei or nuclear condensate fractions were extracted using genomic DNA extraction kit (New England Biolabs). DNA-seq library was prepared from 50 ng of extracted genomic DNA using Illumina Nextera DNA Library Prep kit. DNA-seq experiments were performed in biological replicates. PRO-seq was performed on whole nuclei or nuclear condensate fractions in the storage buffer (Buffer D) as described previously (Kwat et al, 2013), with the following modifications. Briefly, isolated fractions were incubated in the nuclear run-on reaction condition (5 mM Tris-HCl pH 8.0, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.5% Sarkosyl, 0.4 units / μl of RNase inhibitor) with biotin-NTPs and rNTPs supplied (18.75 μM rATP, 18.75 μM rGTP, 1.875 μM biotin-11-CTP, 1.875 μM biotin-11-UTP for uPRO; 18.75 μM rATP, 18.75 μM rGTP, 18.75 μM rUTP, 0.75 μM CTP, 7.5 μM biotin-11-CTP for pChRO) for 5 min at 37°C. Run-On RNA was extracted using TRIzol, and fragmented under 0.2 N NaOH for 15 min on ice. Fragmented RNA was neutralized, and buffer exchanged by passing through P-30 columns (Biorad). 3′ RNA adaptor (/5Phos/NNNNNNNNGAUCGUCGGACUGUAGAACUCUGAAC/3InvdT/) is ligated at 5 μM concentration for 1 hours at room temperature using T4 RNA ligase (NEB), followed by 2 consecutive streptavidin bead bindings and extractions. Extracted RNA is converted to cDNA using template switch reverse transcription with 1 μM RP1-short RT primer (GTTCAGAGTTCTACAGTCCGA), 3.75 μM RTP-Template Switch Oligo (GCCTTGGCACCCGAGAATTCCArGrGrG), 1x Template Switch Enzyme and Buffer (NEB) at 42°C for 30 min. After a SPRI bead clean-up, the cDNA is PCR amplified up to 20 cycles using primers compatible with Illumina Small RNA sequencing.