GSM9290599: PROseq-wtOSC_nuclei_biotin-C; Drosophila melanogaster; OTHER
PROseq libraries were prepared, modified from Mahat et al.(Mahat, Kwak et al. 2016) from both isolated nuclei and permeabilized cells. Cells were harvested, washed with ice-cold PBS, and counted. Nuclei were isolated by resuspension in Douncing buffer (10 mM Tris-HCl pH 7.4, 300 mM Sucrose, 3 mM CaCl₂, 2 mM MgCl₂, 0.1% Triton X-100, supplemented with 0.5 mM DTT, protease, and RNase inhibitors), dounced 25 times with a tight pestle, and pelleted. Permeabilized cells were prepared by resuspension in permeabilization buffer (10 mM Tris-HCl pH 7.4, 300 mM Sucrose, 10 mM KCl, 5 mM MgCl₂, 1 mM EGTA, 0.05% Tween, 0.1% NP40 substitute, supplemented with 0.5 mM DTT, protease, and RNase inhibitors), incubated on ice for 5 minutes, and pelleted. Both nuclei and permeabilized cells were resuspended in storage buffer (10 mM Tris-HCl pH 8.0, 25% glycerol, 5 mM MgCl₂, 0.1 mM EDTA, supplemented with 5 mM DTT), aliquoted, and snap-frozen in liquid nitrogen. Nuclear run-on reactions were performed using 2xNRO buffer supplemented with biotin-11-CTP and biotin-11-UTP (for 2-biotin mixes) or single biotinylated nucleotides (for 1-biotin mixes), ATP, GTP, and RNaseOUT. Samples were incubated at 30°C for 3 minutes, followed by RNA extraction using Trizol-LS and chloroform. RNA was precipitated with Glycoblue and 100% ethanol, incubated for 15 minutes at room temperature, and pelleted by centrifugation (16000 x G, 1 hour, 4°C). Pellets were washed with 75% ethanol and stored at -80°C. Isolated RNA was fragmented by heat denaturation (65°C for 40 seconds) and alkaline hydrolysis (1N NaOH for 10 minutes on ice), then purified using P30 columns. Biotinylated RNA was enriched using MyOne C1 streptavidin beads, washed with high-salt, PROseq binding, and low-salt buffers, and eluted with Trizol and chloroform extraction. RNA was precipitated with Glycoblue and 100% ethanol. After decapping and hydroxyl repair using TAP and PNK enzymes followed by phenol purification, libraries were generated under sRNA seq ligation conditions. 3′ adaptor ligation was performed with T4 RNA ligase 2, truncated KQ, using adaptors containing four random nucleotides at the 5′ end(Jayaprakash, Jabado et al. 2011). After a second streptavidin purification, 5′ adaptor ligation was performed with T4 RNA ligase, using adaptors containing four random nucleotides at the 3′ end, followed by a third streptavidin purification. Reverse transcription was carried out with SuperScript II using an adaptor specific primer. Libraries were PCR amplified with KAPA HiFi polymerase and barcoded Illumina primers in reactions monitored by EvaGreen real time fluorescence. Reactions were stopped after reaching defined RFU thresholds (12–17 cycles). Products were separated on 2.5% agarose gels, gel purified (Zymo), and multiplexed for sequencing (PE50, Illumina HiSeq2500).