GSM6198384: CoLa-seq_rep1; Homo sapiens; OTHER
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).  For each CoLa-seq library, chromatin-associated RNA from 200 million cells was used. To enrich for lariat RNA species (nascent lariat intermediates and excised lariat introns) from the chromatin-associated RNA, RNAs less than roughly 200 nts were removed using SPRI paramagnetic beads (Beckman Coulter) with a beads to sample ratio of 1:1.2. Note that this step also removes excised lariat introns derived from short introns and nascent lariat intermediates derived from short introns with short tails (i.e., short introns that form early during in-order splicing).  Next, ribosomal RNA was depleted using oligo-based Ribo-Zero Magnetic Gold Kit (Illumina) according to the manufacturer's manual. During the optimization of CoLa-seq, we noticed abundant ncRNA reads in our libraries, similar to previous observations in mNET-seq (Mayer et al., 2015). To overcome this issue, biotinylated anti-sense oligos were designed to specifically target and deplete ncRNAs that were found to be abundant in our initial libraries (table S4). Briefly, for every g of rRNA-depleted RNA, a depletion reaction was assembled, containing 82.5 pmol of total depletion oligo mix (table S4), 2X SSC, and 5 mM EDTA. The depletion reaction was put through an oligo annealing cycle (5 min at 75 ˚C, cool down to 22˚C at 0.1 °C/sec), after which ncRNA bound biotinylated depletion oligos were pulled down using Dynabeads™ MyOne™ Streptavidin C1 beads (Invitrogen), according to the manufacturer's manual. The RNA in the supernatant was purified using the RNA Clean and Concentrate kit (Zymo Research) and excess anti-sense oligos were removed by treatment with Turbo DNase (Invitrogen). Finally, according to the manufacturer's instructions, purified RNA was treated with RppH (NEB) to decap linear RNA and then was degraded using Terminator 5'-3' exonuclease (Lucigen). The resulting RNA was put through a size selection step to remove ≤200 nts RNA using SPRI paramagnetic beads (Beckman Coulter) with a beads to sample ratio of 1:1.2. The final RNA quantity is about 0.5-1% of the starting material. Once the chromatin RNA was enriched for lariat species, ~500 ng of RNA was used for CoLa-seq library prep. The 3' ends of RNA were ligated to a pre-adenylated 3' end adapter (table S5), which includes a 6 nt UMI (unique molecule identifier) at the 5' end to allow correction for PCR duplication and RT mis-priming. The ligation reaction contained 17.5% PEG-8000 (w/v), 1X T4 RNA ligase buffer (NEB), 1 µM of the pre-adenylated 3' end adaptor, and 10 units/µL T4 RNA Ligase 2 (truncated K227Q, NEB). The ligation reaction was incubated for 3 hours at 22 ˚C. After the reaction excess adapters were removed by size selection using SPRI paramagnetic beads (Beckman Coulter) with a beads to sample ratio of 1.6:1. Then, the adapter-ligated RNA was used for cDNA synthesis using 0.25 µM of RT primer (table S5) and 10 units/µL of Superscript III (Invitrogen) at 50 ˚C for 1 hour, according to the manufacturer's manual. Afterwards, to degrade excessive RT primer, 3.5 µL of ExoSAP-IT was added to the cDNA synthesis reaction and incubated for another 30 min at 37 ˚C. The reaction was stopped by adding 10 µL of 0.5M EDTA and RNA was degraded by adding 10 µL of 1 N NaOH. The resulting cDNA was cleaned up using Oligo Clean & Concentrator columns (Zymo Research) and was circularized with Circligase (Lucigen), according to the manufacturer's manual. Final library amplification was performed on the circularized cDNA using Phusion polymerase (Thermo Fisher Scientific) with primers that add sequencing adaptor sequences and specific sequencing indices, which are located in the reverse primers (table S5). The number of PCR cycles was between 15 and 18 cycles. The amplified product was run on a 6% TBE gel and DNA fragments between 175 and 750 bps were gel purified and 3 nM of the library was sequenced in a 100 bp paired-end mode on an Illumina HiSeq 4000.