Zea mays B73, 20 min water treatment, primary and seminal root tips (0-1mm), RNA-seq nuclear transcription data, biorep 3
Nuclei were isolated from freshly dissected (0-1mm), paraformaldehyde-fixed B73 root tips of 3-d old seedlings. RNA was isolated from fixed nuclei using Qiagen RNeasy FFPE Kit Reference number 73504 (for paraffin embedded, fixed tissue). We started with Step 6 of Qiagens manufacturers protocol since our samples were not paraffin embedded. The formaldehyde crosslinks are reversed during a 15 min at 80C step and because of the crosslink reversal step, the RNA becomes partially fragmented (RIN ~5). The purified RNA was DNased one time more (the Qiagen kit has a DNase step) with Invitrogen Turbo DNA Free Kit. Ribosomal RNA was removed from the DNase-treated samples using the Qiagen QIAseq FastSelect-rRNA removal kit for plants (Qiagen # 334315). QIA FastSelect was designed to work with existing RNA-seq next generation library preparation kits so we followed the QIAseq FastSelect-rRNA removal kit instructions as it pertained to making libraries using the NEBNext Ultra II Directional RNA Library kit for Illumina (NEB # E7760S). We used a mass input of 1 ug of nuclear RNA in a total volume of 4 ul and mixed it with 4 ul of the NEBNext First strand Synthesis reaction buffer, 1ul of the NEB random primers and 1 ul of the QIAseq FAstSelect rRNA plant reagent. We then followed the QIAseq FastSelect guidelines for partially degraded RNA (RIN 2-6) for the simultaneous fragmentation (15 min at 94 C) of RNAs and hybridization of the QIAseq FastSelect reagent to RNAs. We then immediately proceeded to the First Strand cDNA Synthesis Reaction (Step 4.2) of the NEBNext Ultra II Directional RNA Library Prep instruction manual and followed all subsequent steps targeting 200-nt inserts. At the adaptor ligation step, we used the NEBNext Unique Molecular Indicators (UMI) and Unique Dual Indice adapter kit Ref #E7416S with no adaptor dilution. The adaptor ligated DNAs were amplified for 7 PCR cycles, had an average library size (insert + 150 bp adaptor) ranging from 395-398 bp, making the average sequenceable insert 245-248 bp.