GSM7863730: Native RNA from Rbp3 immunoprecipitated material size selected 50-170nt; Saccharomyces cerevisiae; OTHER
Size selected NET-Seq was developed to map new transcription units and their putative transcription start sites (TSS) on the S.cerevisiae genome. The step up to ligation of linker-1 were performed as for the conventional NET-Seq protocol (Fischl et al., 2017; http://dx.doi.org/10.1016/j.molcel.2017.01.006); Uzun et al., 2021;  http://dx.doi.org/10.1016/j.celrep.2021.109755). For size selected NET-Seq, 6ug of RNA (>3ug) was used and split into six non-stick RNase-free tubes for the ligation reaction, just as for conventional NET-Seq. After 3 hours incubation at 37°C, the reaction was quenched by adding 0.7ul of 0.5M EDTA to each tube. The ligation product was purified and concentrated by isopropanol precipitation. Specifically, 0.56ml of RNA solution and 0.75ml of isopropanol were added to each tube. After vortexing, the mixture was incubated at ~20°C for 2 hours, followed by centrifugation at 4°C, 20,000g for 30mins. Next, the supernatant was removed without disturbing the RNA pellet. The pellet was then washed once with 0.75ml of 80% ice?cold ethanol. After brief centrifugation, residual ethanol was carefully removed by pipetting. The pellet was air-dried at room temperature for 10mins. The RNA pellets were resuspended in 15ul of 10mM Tris.Cl (pH 7.0), mixed with 15ul of 2X TBU denaturing loading buffer, denatured at 80°C for 3 mins, and 10ul loaded onto 3 wells of an ice-cold 3.5% TBU polyacrylamide gel (10mls 4.8g Urea, 1.17ml 30% acrylamide solution, 50uL 10% APS, 1 mL 10X UltraPure TBE buffer, MilliQ water to 10mls, 10uL TEMED poured between glass plates cleaned with 1% SDS and washed thoroughly with MilliQ water to remove RNase contamination. The gels were left for one hour to set and then pre?run for 15mins. Before loading, a pipette with a clean tip was used to flush the wells with 1XTBE buffer several times. Two empty wells were left between the ladders (heated denatured 10bp, 50bp, 100bp and 1kb DNA ladders) and the sample. The samples were subject to electrophoresis using ice-cold 1xTBE running buffer, at 80V for 80mins. The gel was stained for 5mins in 50ml of 1xTBE buffer containing 5ul of SYBR Gold nucleic acid gel stain (S11494, ThermoFisher Scientific), followed by destaining in 1xTBE buffer for another 5mins. The gel was frozen at ~20°C for 15mins, prior to cutting into size ranges, normally 20-50nts (samples A), 50-170nts (samples B), 170-500 (samples C) and 500-1000nts (samples D). Sample E are fractions >1000nt but did not yield much beyond rRNA sequences. The RNA was recovered from mashed gel pieces using electroelution in 1 x TBE at 80V with the electrophoresis tank sitting in wet ice. Fluorescence was monitored every 10mins with the transilluminator, with 10mins being sufficient for the smallest fraction and up to 2hrs for the longest RNA.  After electroelution, RNA from each size fraction was dissolved in 600ul of 10mM Tris.Cl (pH 7.0). To purify and concentrate the RNA, 2ul of Glycoblue, 50ul of 3M sodium acetate (pH 5.5) and 0.75ml of isopropanol were added. The RNA pellet of the smallest fraction was dissolved in 10ul of 10mM Tris.Cl (pH 7.0). All the other size fractions were dissolved in 20ul of the same buffer. The RNA from each fraction, except the smallest, is subject to alkaline fragmentation and library construction, with S.pombe NET-Seq spike-in, following the conventional NET-Seq protocol (Fischl et al., 2017; http://dx.doi.org/10.1016/j.molcel.2017.01.006); Uzun et al., 2021;  http://dx.doi.org/10.1016/j.celrep.2021.109755), except that up to 2 more PCR cycles may be needed. The sequencing of NET-Seq libraries was performed on the Illumina NEXTSeq 500 Sequencer with the high throughput flow cell, single end reads and 50 cycles. NET-Seq normally requires 100 million reads to obtain good coverage on each S.pombe spiked S.cerevisiae sample. NET-Seq nascent RNA sequencing libraries based on protocols in Fischl et al., 2017; http://dx.doi.org/10.1016/j.molcel.2017.01.006; Uzun et al., 2021;  http://dx.doi.org/10.1016/j.celrep.2021.109755 in