GSM9702740: alpha-amanitin 6h rep4; Arabidopsis thaliana; OTHER
RNA was isolated according to a standard seed protocol (https://doi.org/10.1002/biot.200900211). 500 ng of seed RNA was used for reverse transcription (2.5 ul of 1000x-diluted ERCC spiked into each sample) with 50 mM of various barcoded oligo(dT) primers and SuperScript III. 0.25 of each reaction was 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).