GSM5852164: WB_pChRO_EK54_2003; Homo sapiens; OTHER
For chromatin preparation, 1 ml of frozen blood sample is thawed and lysed in 10 ml of the NUN buffer (0.3M NaCl, 1M Urea, 1% NP-40, 20mM HEPES, pH 7.5, 7.5mM MgCl2, 0.2mM EDTA, 1x protease inhibitor cocktail, 1 mM DTT, 4 u/ml RNase inhibitor) with gentle mixing. Blood chromatin is pelleted by centrifugation at 15,000 g for 20 min, 4°C, and resuspended in Wash buffer (50 mM Tris pH 7.5). After brief centrifugation, the pellet is washed once more in Buffer D (50 mM Tris-HCl, pH 8.0, 25% glycerol, 5 mM Mg Acetate, 0.1 mM EDTA, 5 mM DTT), then homogenized using short sonication cycles in 50 μl of Buffer D. For peripheral leukocyte isolation, 5-10 ml of peripheral whole blood is sampled from brachial veins. A final concentration of 1.5 mM EDTA is added to the whole blood to prevent clotting. To isolate PBMC and PMNL from the peripheral blood, 3 ml of Poly Cell Separation Media, 4 ml of Human Cell Separation Media, and 5 ml of blood are carefully layered consecutively with minimal mixing. Separation occurs with a centrifugation of 450-500 g for 30-35 minutes at 18-22°C. 3 interface layers should be visible. The top interface is PBMC and the middle interface including the layer right below is PMN. 5ml of each PBMC and PMN layers are collected in 1.7 ml microcentrifuge tubes. PBMC and PMN are pelleted by centrifugation at 4,000 for 4 min, 4°C, and washed in PBS. Centrifugation and wash is repeated once more. After brief centrifugation, the pellet is washed in Buffer D (50 mM Tris-HCl, pH 8.0, 25% glycerol, 5 mM Mg Acetate, 0.1 mM EDTA, 5 mM DTT), then resuspended in 50 μl of Buffer D. Chromatin or cells 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. smallRNA-seq