GSM4190169: K562_DMSO_rep1 [TIF-seq2]; Homo sapiens; OTHER
RNA was extracted with Trizol and Direct-zol RNA kit from Zymo Research. TIF-Seq2: 2.5 µg total DNase treated RNA was dephosphorylated using Calf Intestinal Alkaline Phosphatase (CIP; NEB). Sample was phenol-chloroform purified and precipitated with ethanol. mRNA was decapped by incubating with Cap-Clip (CellScript) at 37˚C for 60min. After phenol-chloroform purification and ethanol precipitation, RNA was ligated overnight at 16˚C at 5' with DNA/RNA oligonucleotide adaptor (TIF2-RNA: TCAGACGTGTGCTCTTCCGATCTrNrNrWrNrNrWrNrN) using T4 RNA ligase (NEB) in the presence of 10% DMSO, RiboLock RNase Inhibitor and 1mM ATP. Ligated RNA was purified with 1.8X Ampure XP beads according to the manufacturer instruction and then used as the template for reverse transcription. Ligated mRNAs were reverse transcribed using barcoded oligo-dT primers (TIF2-RT: TAGTTCAGTCTTCAGTACCTCGTGCGGCCGCXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCTTTTTTTTTTTTTTTTVN; where X refer to a specific barcode). For the revers transcription reaction, mRNA was mixed with the corresponding TIF2-RT oligo and dNTPs, denatured at 65˚C for 5min and then put on ice. The sample was mixed with first strand buffer, Trehalose and RiboLock RNase inhibitor and incubated first at 42˚C for 2 min. Finally 2 µl of SuperScript III (Thermo Fisher Scientific) was added to each reaction and incubated at 42˚C for 50 min, at 50˚C for 30 min, at 55˚C for 30 min and inactivated at 70˚C for 15 min. RNA template was removed by incubating with RNase H and RNase cocktail (Ambion) at 37˚C for 30min. First-strand cDNA was purified with 2X Ampure XP beads. To avoid saturating the reaction, only half of the obtained cDNA was used for the following PCR, and the rest was stored as a backup. Used cDNA template was further split into two PCR reactions with Terra PCR Direct Polymerase (Takara) with the following program 98˚C for 2min, then 16 cycles of 98˚C for 20s, 60˚C for 30s, 68˚C for 5min (+10s/cycle) and finally 72˚C for 5min. The PCR above used as primers TIF2-Rv: TAGTTCAGTCTTCAGTACCTCGT and TIF2-Fw: TATAGCGGCCGCXXXXXXGTGAC[BtndT]GGAGTTCAGACGTGTGCTCTTCCGATC (where X refers to different barcodes). PCR products were purified with 1X Ampure XP beads and quantified with qubit dsDNA HS assay. PCR products from different samples were pooled together with equal mass. Pooled PCR products were subjected to NOT1-HFNEB) digestion at 37˚C for one hour. After inactivation of NOT1 at 65˚C for 20 min, samples were purified with 1X Ampure XP beads. To favour intramolecular ligation, PCR products were diluted to a final concentration less than 1ng/µl and ligated with a high concentration of T4 DNA ligase (NEB) at 16˚C for at least 16 hours. Unligated linear PRC products were removed by treatment with plasmid-safe. After inactivation at 70˚C for 30min, the circularized cDNA was purified with phenol-chloroform and ethanol precipitation. Circularized cDNA was fragmented by sonication (Covaris ME220; Covaris, Inc) (Duration 240s, peak power 30, duty factor 10, Avg Power(W) 6). The fragments were then purified with 1X Ampure XP beads. Biotin labelled fragments which contained the 5' and 3' connecting region was enriched using Dynabeads M280 streptavidin (Invitrogen) and incubating at room temperature for 30 min. Captured fragments were subjected to end repair and dA tailing. To add the required Illumina grafting sequences, each sample (20.5 µl beads) was incubated at room temperature for one hour with a mixture of 10 µl 5X quick ligation buffer (NEB), 19.5 µl nuclease-free water (Ambion), 3 µl T4 DNA ligase and 1 µl 1µM duplexed adaptors. Duplex adaptors where generated by annealing TIF2-forkFw (5'-AATGATACGGCGACCACCGAGATCTACACACACCTGCCGGTCACC*T-3) and TIF2-forkRv (5'-phos-GGTGACCGGCAGGTGTATCTCGTATGCCGTCTTCTGCTTG-3') at 1µM. After cleaning the beads, the beads were resuspended in 20µL H2O and used for PCR amplification using 25µl Phusion High-fidelity MasterMix (2X) (NEB), 4 µl nuclease-free water and 1 µl PCRgraftP5 primer (10µM, 5'-AATGATACGGCGACCACCGAGATCTACAC-3') and 1 µl PCRgraftP7 primer (10µM, 5'- CAAGCAGAAGACGGCATACGAGAT-3'). Samples were subjected to PCR amplification with the following program: 98˚C for 30s, then 18 cycles of 98˚C for 20s, 65˚C for 30s, 72˚C for 30s and finally 72˚C for 5min. DNA library then went through two step beads-based size selection (first step 0.35X and second step 0.45X) with expected size distribution in the range of 300bp-1000bp. Purified DNA library was adjusted to 4nM, then denatured, diluted according to Illumina instruction and sequenced on Nextseq 500 platform. We used four custom sequencing oligos as follows: SeqR1+15T (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTTTTTTTTTTTTTT-3'), SeqINDX1 (5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3'), SeqINDX2 (5'-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3') and SeqR2 (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'). Two mixtures of sequencing oligos were prepared as follows: mixture 1 contained SeqR1+15T(0.3µM) and SeqR2(0.3µM), mixture 2 composed of SeqINDX1 primer (0.3µM) and SeqINDX2 primer (0.3µM). Mixture 1 was loaded into both positions (#7 and #8 on reagent cartridge in a NextSeq 500 instrument) for custom read1 primer and for custom read2 primer. Mixture2 was loaded into the position for custom index primer (#9 on reagent cartridge). Sequencing was carried out in an Illumina NextSeq 500 instrument with stand-alone configuration and custom sequencing oligos. Paired end sequencing read lengths were set as read1 76bp, read2 76bp, index1 6bp and index2 6bp.
GEO Accession
GSM4190169