GSM4083795: F19; Homo sapiens; RNA-Seq
Cells (approximately 0.1 g wet weight) were lysed on ice (5 min) by resuspension in Pierce IP Lysis Buffer (0.8 mL; 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol; Thermo Fisher) containing 1x protease inhibitor cocktail III (Calbiochem). The resulting lysate was clarified (17,000g, 10 min, 4 °C) and left at room temperature (30 min). The sample was filtered (Ultrafree-MC filter unit (Millipore); 12,000g, 2 min, 4 °C) to remove insoluble aggregates. All lysates were subject to size exclusion chromatography (SEC) using an Agilent 1100 HPLC system (Agilent Technologies, ON, Canada) with a multi-phase chromatography protocol as previously described (Havugimana et al., 2012). Fractions from a biological replicate SEC separation corresponding to higher molecular weight species (approximately >1.5 MDa; fractions 16-23 in Figure 1B) were analyzed by total RNA sequencing. Total RNA was isolated from each fraction (0.375 mL) by addition of Trizol (1.125 mL; Thermo Fisher) and the sample (1.4 mL) was transferred to a Phasemaker tube (Thermo Fisher). Total RNA was extracted following the protocol supplied by the manufacturer and further cleaned up using a RNeasy MinElute Cleanup Kit (Qiagen). RNA integrity number (RIN) was measured using an Agilent Bioanalyzer and samples were ribo-depleted using a RiboZero Gold (Human/Mouse/Rat) kit (Illumina) to remove rRNAs. RNA libraries were prepared for sequencing according to vendor protocols using NEBNext R Small RNA Library Prep Set for Illumina R (Multiplex Compatible), Cat #E7330L, according to the protocol described by Podnar et al. (Podnar et al., 2014). RNA was fragmented using elevated temperature in carefully controlled buffer conditions to yield average fragment sizes of 200 nucleotides. These fragments were directionally ligated to 5' and 3' adaptors so that sequence orientation is preserved throughout sequencing. Reverse transcription and PCR were performed to complete the DNA sequencing libraries, which were sequenced using an Illumina NextSeq 500 instrument (75-nt single reads) at the Genomic Sequencing and Analysis Facility at the University of Texas at Austin.