title: GSM6198392: lariat-seq_rep1; Homo sapiens; OTHER
library_construction_protocol: Chromatin RNA was isolated from K562 cells similar to previously described methods (Pandya-Jones and Black, 2009; Wuarin and Schibler, 1994; Werner and Ruthenburg, 2015), with modifications noted below. All the steps were performed with pre-chilled, ice-cold, freshly prepared RNase-free buffers using RNase-free equipment on ice or at 4 ˚C. Roughly 5% of subcellular fractions were saved for quality control (western and RNA fragment size analysis). Fifty million K562 cells were spun down at 100 x g for 3 minutes and washed twice with 15 mL of ice-cold 1X PBS (phosphate-buffered saline) and once with 1 mL of ice-cold 1X PBS. Washed cells were then gently resuspended in 1mL of 0.1% (v/v) Triton X-100-containing buffer A (10 mM HEPES (pH 7.5), 10 mM KCl, 10% glycerol, 0.116 g/mL sucrose, 1 mM DTT, 1x protease inhibitor, 2 mM EDTA) and lysed on ice for 9 min. After incubation, the lysed cells were centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant corresponding to the cytoplasmic fraction was carefully removed. The remaining nuclei pellet was gently resuspended in 250 µL of NRB buffer (20 mM HEPES (pH 7.5), 75 mM NaCl, 50% glycerol, 0.5 mM EDTA, 1 mM DTT, 1x protease inhibitors). Then, 250 µL of NUN buffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 0.2 mM EDTA, 1 M urea, 1 mM DTT, 1% NP-40) was slowly added and mixed by inversion. The resulting nuclei suspension was incubated on ice for 5 minutes and centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant corresponding to the nucleoplasmic fraction was carefully removed. The chromatin pellet was washed once with 500 µL of premixed NRB/NUN buffer (1:1) and centrifuged at 800 x g for 5 minutes at 4 ˚C. The supernatant was carefully removed. The washed chromatin pellet was gently resuspended in 200 µL of NRB buffer. The chromatin pellet were extracted by Trizol (Invitrogen) followed by a second extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and isopropanol precipitation. The isolated RNA was furthered treated with Turbo DNase (Invitrogen) to remove residual genomic DNA, followed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1). 10 µg chromatin-associated RNAs were depleted of ribosomal RNA depletion using Ribo-Zero Magnetic Gold Kit (human/mouse/rat; Illumina), according to the manufacturer's instruction. To further enrich lariat RNA species, RNase R (Lucigen) digestion was performed to degrade linear RNA, according to the manufacturer's instruction; treatment also trims tails of lariat RNA species. The resulting RNA was used to prepare an RNA-seq library using NEBNext Ultra™ Directional RNA Library Prep Kit from Illumina, according to the manufacturer's instruction except for the RT step, during which 1 mM MnCl2 was included to facilitate reverse transcriptase go through the BP. The final library was sequenced in a 100 bp paired-end mode on an Illumina Hiseq 4000.