title: GSM8547567: IVT mRNA, input2, rep#2; Homo sapiens; OTHER
library_name: GSM8547567
library_construction_protocol: Generally, with harvesting cells as the first step, the media was aspirated, and the cells were washed once with proper volume of ice cold DPBS buffer for each plate. Then total RNA was isolated from cells with TRIzol reagent (Invitrogen) and then extracted following manufacturer's protocol by isopropanol precipitation. Small RNA fraction was purified from total RNA with RNA Clean & Concentrator kit (Zymo Research) following manufacturer's protocol. mRNA was extracted by two rounds of polyA+ purification with Dynabeads mRNA DIRECT kit (Ambion). CRACI for D site detection Wild-type HepG2, mESC, and arabidopsis thaliana seedling cells (two replicates for each sample, one 10-cm plate per replicate); HepG2 siControl or DUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate). After extracting the polyA+ RNA or small RNA from mammalian cells or plant cells, around 100 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with Oligomer Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by Oligomer Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water.  To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (5′App-NNNNNATCACG AGATCGGAAGAGCACACGTCT-3SpC3, with ATCACG as the inline barcode) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 45 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 45 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 10 µl RNase-free water. The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUC -3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 12 µl RNase-free water.  A 2 µl aliquot of the purified RNA was saved for 'Input1' library construction, 2 µl aliquot of the purified RNA was saved for 'Input2' library construction, and the rest 6 µl was subjected to CRACI optimized reduction treatment, as the 'Treated' sample. The 6 µl RNA was mixed with 40 µl freshly prepared KBH4 (1 M KBH4, prepared by dissolving 54 mg KBH4 in 1000 µl RNase-free water) and Incubated the reaction mixture at 25 °C for 3 h, followed by purification with RNA Clean and Concentrator (Zymo Research) and elute with 10 µl H2O. 'Input1', 'Input2' and purified 'Treated' samples were diluted to 10 µl and mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. Then for 'Input2' and 'Treated' samples, a stock with 2 µl 10x AMV RT Buffer (New England BioLabs, cat. no. B0277AVIAL), 2 µl 1 mM dNTP Solution Mix (dilute from 10 mM dNTP Solution Mix, New England BioLabs, catalog no. N0447L), 2 µl 10mM dGTP (dilute from 100 mM dNTP Solution Mix, New England BioLabs, catalog no. N0442S), 1 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 2 µl HIV RT (Worthington Biochemical, catalog no. LS05003) was added. For 'Input1' sample, a stock with 2 µl RNase-free water, 2 µl 10x AMV RT Buffer, 2 µl 10 mM dNTP Solution Mix (New England BioLabs, catalog no. N0447L), 1 µl RNaseOUT Recombinant Ribonuclease Inhibitor and 2 µl HIV RT was added.  The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina NovaSeqX with single-end 100 bp read length.