GSM8516397: silique; treated; replicate 1; Arabidopsis thaliana; OTHER
Total RNA isolation: total RNA was isolated according to TRIzol reagent manufacturer's protocol, followed by isopropanol precipitation. PolyA+ RNA isolation: Dynabeads™ mRNA Purification Kit (Invitrogen™) was used for polyA+ RNA enrichment, from the purified total RNA. With 10 ng synthetic RNA oligos carrying 0%, 20%, 40%, 60%, 80%, or 100% pseudouridine, 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). To perform 3′-adapter ligation, 10 µl 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (5′App-NNNNN 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 30 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 30 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 9.3 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 10 µl RNase-free water.  A 1.5 µl aliquot of the purified RNA was saved for 'Input' library construction, 8.5 µl was subjected to BID-seq optimized bisulfite treatment, as the 'Treated' sample. The 8.5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 900 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 75 µl RNase-free H2O, 270 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Then the 'Input' was diluted to 10.5 µl with RNase-free water. Both 'Input' and 'Treated' samples were 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. To this was added 4 µl 5× SSIV Buffer, 2 µl 10 mM dNTP Solution Mix (NEB), 1 µl 100 mM dithiothreitol, 0.5 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 1 µl SuperScript IV Reverse Transcriptase (SSIV, catalog no. 18090050, Thermo Scientific). The reaction was mixed well and incubated at 50 °C for 1 h, followed by adding 1 µl RNase H (catalog no. M0297L, NEB) and incubating at 37 °C for 20 min. 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 Nova-seq 6000 with single-end 100 bp read length.