GSM6198396: direct-RNA-pulldown_rep2; Homo sapiens; OTHER
Total RNA were extracted from K562 cells using Trizol (Invitrogen) followed by a second extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and isopropanol precipitation. The isolated total RNA was furthered treated with Turbo DNase (Invitrogen) to remove residual genomic DNA, followed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1). Then, 1 mg of total RNA was incubated with three sets of biotinylated anti-sense oligos (ASOs), which target intron 16 of HNRNPK,  intron 1 of EIF, and intron 6 of PPP2CA (1 µM per oligo; Table S7), in a solution containing 2x SSC, 5 mM EDTA, and 10% Formamide. The resulting hybridization reaction was put through an oligo annealing cycle (5 min at 80 ˚C, cool down to 22˚C at 0.1 °C/sec), after which RNA bound biotinylated ASOs were pulled down using Dynabeads™ MyOne™ Streptavidin C1 beads (Invitrogen), according to the manufacturer's manual. The ASOs were digested with DNas I and the pulled-down RNA was extracted using Trizol and precipitated by ethanol. 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 (Genomics Facility at University of Chicago).