GSM4190170: K562_DMSO_rep1 [3T-fill]; Homo sapiens; OTHER
RNA was extracted with Trizol and Direct-zol RNA kit from Zymo Research. 3'T-fill: We performed 3'T-fill as previously described (PMID: 23295673). In brief, 10µg DNA-free total RNA (16µL) was fragmented by adding 4µl fragmentation buffer(5X)(200mM Tris-acetate, pH 8.1, 500mM Potassium Acetate, 150mM Magnesium acetate) and incubated at 80˚C for 5min. Fragmented RNA was purified with 1.5X Ampure XP beads and used as the template for reverse transcription(RT). Sample were mixed with biotinylated dT primer (P5_dT16VN: 5'- [Btn]AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTTTTTTTTTTTTTTVN-3') (where V refers to A, C or G) and denatured at 65˚C for 5min. Denatured RNA was then mixed with 4µl 5X first strand buffer, 2µl DTT(0.1M) and 4µl freshly prepared actinomycin D(0.1µg/ul). After incubation at 42˚C for 2min, 0.5µl SuperScriptII (Invitrogen) was added, incubated at 42˚C for 50 min and finally 72˚C for 15min. cDNA was purified with 1.5X Ampure XP beads according to manufacturer instruction. For second strand synthesis, cDNA was mixed with of 0.5 µl RNase H (5U/µl)(NEB) and 2 µl DNA Polymerase I (10U/µl) (Thermo Scientific) and incubated at 16˚C for 2.5 hours. cDNA was purified with 0.9X Ampure XP beads according to manufacturer instruction. 20 µl biotin labelled cDNA was mixed with 20µl Dynabeads M280 streptavidin beads (Invitrogen) at room temperature for 15 min. Following end repair and dA tailing, each sample(8 µl) was mixed with 12.5 µl 2X Quick Ligation buffer (NEB), 2.5µL T4 DNA ligase (2000U/µL, NEB) 2µl of anneal duplexed adaptors at 2.5 µM(5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3', 5'-Phos-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[AmC7]-3') and incubated for at 20˚C for 20min. Adaptor ligated libraries were washed and amplified by PCR by adding 25 µl 2X Fusion High Fidelity Master Mix, 0.5 µl PE1 primer (10µM, 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3')and 0.5µl PE2_MPX primers (10µM, 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3'; where X refer to the specific barcode). Samples were subjected to the following program: 98˚C for 30 seconds, 18 cycles of 98˚C for 10s, 65˚C for 10s, 72˚C for 10s and finally 72˚C for 5min. Library was purified with 1.8X Ampure XP beads according to manufacturer instruction. 3´T-fill libraries were sequenced simultaneously with TIF-Seq2 libraries (using same condition and sequencing oligonucleotides).
GEO Accession
GSM4190170