GSM5452295: PL_size_selected_SNU_WT; Homo sapiens; RNA-Seq
Nascent, thio-labelled RNA was generated as described for SNU-Seq without size selection. The nascent RNA was then run on a 3.5 % TBE-Urea gel (8M urea). For this, 250 ng of nascent RNA was mixed with 2x loading dye and boiled for 2 minutes at 80 ◦C. The gel was kept on ice and pre-run for 10 minutes at 80 V, using ice-cold ultra-pure TBE (1x) as the running buffer. Wells of the gel were resuspended with TBE before loading samples. Samples were then run at 80 V for 90 minutes. The gel was transferred onto a square petri dish and incubated in 1x ultra-pure TBE with SYBR Gold (1/10,000 v/v) for 5 minutes. The gel was rinsed twice in 1x ultra-pure TBE before imaging.  For purification of the small nascent RNA fraction, the gel was incubated at -80◦C for 10 minutes. The small band of 70-100 bp size was cut using a razor blade. RNA was extracted from the gel slice using dialysis: The gel slice, along with 0.8 mL 10 mM Tris-HCl (pH 7), was placed into SnakeSkin dialysis membrane sealed by two Eppendorf caps. The dialysis was run for 35 minutes at 80 V. The RNA was then purified with isopropanol precipitation and eluted in 11 µL RNase-free water. Quality and size distribution of the size-selected RNA was checked on the Agilent Bioanalyzer using an RNA pico chip.   The size selected nascent RNA was treated with 5' Pyrophosphohydrolase (NEB) in ThermoPolQR reaction buffer following the manufacturer's instructions. The reaction was stopped with 1 µL 500 mM EDTA and heat-inactivated by incubation at 65 ◦C for 5 minutes. RNA was again purified with isopropanol precipitation and resuspended in 6 µL RNase-free water. The NEBNext Small RNA Library kit for Illumina following the kit instructions. Library quality was assessed with the Agilent Bioanalyzer using a DNA High Sensitivity chip. Sequencing was performed on an Illumina NextSeq 500 platform. Thiolabelled spike-ins were generated by amplifying 3 1000-bp long DNA frag- ments from genomic yeast DNA with a 42 % GC content, covering the genes bat2, hxt1, and gal1 using forward primers containing the T7 promoter sequence. The PCR product was purified and then transcribed in vitro following the MEGAscript T7 tran- scription kit instructions, using a thio-UTP to UTP ratio of 1:5.