GSM9702688: input 6h imbibition rep4; Arabidopsis thaliana; OTHER
RNA was isolated according to a standard seed protocol (https://doi.org/10.1002/biot.200900211). For 4-SU labelled RNAs enrichment, biotinylation was performed for 16 μg of freshly isolated RNA in a 200 μl reaction mixture (10 mM Tris pH 7.5; 1 mM EDTA; 0.2 mg/ml EZ-Link Biotin-HPDP (ThermoFisher)) for 1.5 h at 22°C. 1 µl of tenfold diluted ERCC RNA Spike-In Mix (ThermoFisher) and in vitro transcribed 4-SU labelled transcripts were added to the RNA before the biotinylation reactions, and a portion of the RNA after the addition of spike-ins (500 ng) was retained as an input. Unbound Biotin-HPDP was removed by chloroform/isoamyl alcohol (24:1) extraction. Subsequently, a 1/10 volume of 5 M NaCl and an equal volume of isopropanol were added, and RNA was precipitated by centrifugation at 20,000g for 20 minutes. The pellet was washed with 1 ml of 80% ethanol, and after drying, it was resuspended in 25 μl of RNase-free water. Biotinylated RNA was purified using 50 μl of streptavidin C1 Dynabeads (ThermoFisher). The beads were washed in binding buffer (0.2 M NaCl; 0.1 M sodium phosphate pH 6.8; 25 mM MgCl2; 0.4% Tween), blocked in the binding buffer with 1 μg/μl glycogen for 10 minutes, and washed again in binding buffer. RNA samples were denatured at 65°C for 5 minutes, followed by 5 minutes on ice. Then, 25 μl of 2x binding buffer and 50 μl of washed streptavidin beads were added, mixed, and incubated at 22°C with gentle shaking for 20 minutes. The beads were washed three times with 0.5 ml of washing buffer (100 mM Tris-HCl pH 7.5; 10 mM EDTA; 1 M NaCl; 0.1% Tween20) at 65°C, followed by three washes at room temperature. Biotinylated RNA was eluted from the beads by two incubations with 50 μl of 200 mM DTT. RNA from the combined eluates was purified using RNA Clean & Concentrator-5 columns (Zymo), following the protocol for long RNA purification with DNase treatment. The entire volume of the sample was used for reverse transcription. Input and enriched RNA samples were used in reverse transcription with 50 mM of various barcoded oligo(dT) primers and SuperScript III. 0.25 of each input and whole enriched RNA RT reaction were pooled. cDNA pools were purified with Ampure beads (1.5x beads to sample volumes) and then eluted in 17 µl of water. 15 µl of the recovered cDNA pools were mixed by pipetting with 15 µl of the second-strand synthesis mix (2x NEBNext Second Strand Synthesis (dNTP-free) Reaction Buffer (NEB), 1 U RNaseH (NEB), 1 U E. coli DNA ligase I (NEB), 5 U E. coli DNA polymerase (NEB), 30 µM dNTPs) and incubated overnight at 16°C. The reactions were purified with Ampure beads (1.2x beads to sample volumes) and eluted in 4 µl of water. 3 µl of the recovered dsDNA samples were used for tagmentation with 3 µl of 5x diluted B-loaded Tn5 enzyme (homemade) and 6 µl of freshly prepared 2x buffer (20 mM Tris-HCl pH 7.5, 20 mM MgCl2, 50% DMF). The reactions were mixed by pipetting and incubated for 7 minutes at 55°C, followed by 5 minutes at 80°C. Water was added to a total volume of 36 µl; samples were purified with Ampure beads (0.85x beads to sample volumes) and eluted with 21 µl of water. Illumina indexing PCRs were performed using 20 µl of the tagmented DNA in a 50 µl reaction with Q5 2× PCR Master Mix (NEB) and a final concentration of 0.5 µM of primers. Libraries were sequenced on the Illumina Nova-seq to obtain two reads: R1 (containing barcode and UMI) and R2 (containing mRNA sequence).