GSM6533007: Sample 106_HeLa-WT_polyA-RNA_Input_rep3; Homo sapiens; RNA-Seq
Total RNA isolation: cellular total RNA was isolated according to TRIzol reagent manufacturer's protocol, followed by isopropanol precipitation. PolyA+ RNA isolation: Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen™) was used for polyA+ RNA enrichment, from the purified total RNA. Wild-type HeLa, HEK293T or A549 cells were prepared by 3.5-cm plate per replicate. After extracting the polyA+ RNA from cultured cells, ~10-20 ng RNA was fragmented using RNA Fragmentation Reagents (Invitrogen) and heated at 70 °C for 14 min, followed by purification with RNA Clean & Concentrator (Zymo Research). 3'-end repair and 5'- phosphorylation were conducted with T4 Polynucleotide Kinase (EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL of 10X T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB) and 3 µL of T4 PNK, diluted to a final volume of 30 µL, and incubated at 37 °C for 45 min; then 1.5 µL of T4 PNK and 1.5 µL 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by RNA Clean & Concentrator purification eluting with 7 uL RNase-free water. To perform 3'-adaptor ligation, 6 µL 3'-repaired and 5'-phosphorylated RNA fragments were incubated with 1.0 µL 3'-Adaptor (5'App-NNNNNATCACGAGATCGGAAGAGCACAC GTCT3SpC3, with ATCACG as the inline barcode) at 70 °C for 2 mins and immediately placed on ice. 10 µL 3'-Ligation Reaction Buffer (2X) (NEB) and 3 µL 3'-Ligation Enzyme Mix (NEB) were added to the RNA-adapter mixture. The reaction was incubated at 16 °C for 12 hrs. Then 1.0 µL SR RT Primer for Illumina (NEB) was added to the mixture, followed by 5 min at 75 °C, 15 min at 37 °C, 15 min at 25 °C, and then on ice. The denatured 1.0 µL 5'-Adaptor (5'- GUUCAGAGUUCUACAGUCCGACGAUCNNNNN-3') was added into the mixture, together with 1.0 µL 5'-Ligation Reaction Buffer (10X) (NEB) and 2.5 µL 5'-Ligation Enzyme Mix (NEB). The reaction was incubated at 25 °C for 12 hrs, followed by RNA Clean & Concentrator purification, eluting with 10 µl RNase-free water. 1 µL of the purified RNA was saved for “Input” library construction, 9 µL was subjected to BID-seq optimized bisulfite treatment, as the “Treated” technical replicates. For each replicate, 9 µl RNA was mixed with 45 µL freshly prepared BS reagent and incubated at 70 °C for 4 h, then 25 uL 1.5 M Tris buffer 8.8 was added and incubated at 37 °C for 1 h for desulphonation. Most of the bisulfite reagent was removed by running BioRad P6 column and the eluent was further 22 purified by RCC column from Zymo, eluting with 12 uL RNase-free water. Then the “Input” was diluted to 12 µL with RNase-free water. Both the “Input” and “Treated” samples were mixed with 4 µL 5X SSIV Buffer (Thermo Scientific), 1 µL of 10 mM dNTP Solution Mix (NEB), 1 µL 100 mM DTT, 1 µL of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Scientific), and 1 µL of SuperScript IV Reverse Transcriptase (Thermo Scientific). The reaction was and incubated at 50 °C for 1 hr. 1.0 µL of the reaction mixture was used to conduct qPCR to determine the PCR cycle numbers. Then 10 µL cDNAs were amplified using the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina) and purified by agarose gel. All libraries were sequenced on Illumina Nova-seq 6000 with single-end 100 bp read length.