title: GSM9040826: Zea mays B73, primary and seminal root tips (0-1mm), 2 h water treatment, RNA-seq cellular transcription data, biorep 3; Zea mays; ssRNA-seq
library_construction_protocol: The terminal 1mm root tip tissue tissue was ground with mortar and pestle in liquid nitrogen and RNA was extracted using TRIzol from Invitrogen #15596026. DNA was removed from the RNA using the 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 FasSelect was designed to be incorporated into the library preparation kit protocols. Please see the library construction protocol  for details. RNA-seq libraries were made using the NEBNext Ultra II Directional RNA Library kit for Illumina (NEB # E7760S) in combination with NEBNext Multiplex Oligos for Illumina, Unique Dual Index UMI Adaptors RNA set 1 (NEB # E7416S) and the Qiagen QIAseq FastSelect plant ribosomal RNA removal kit. Immediately prior to the library construction, 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. Per the QIAseq FastSelect guidelines, we used a mass input of 1 ug of total cellular 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 guidlelines for intact RNA (RIN > 7) (See Table 7 of the Qiagen kit manual) for the simulatneous fragmentation (15 min at 94 °C) of RNAs and hybridization of the QIAseq FastSelect reagent to RNAs. We then immediately proceeded to the “First Sstrand cDNA Ssynthesis Reaction” (Step 4.2) of the NEBNext Ultra II Directional RNA Library Prep instruction manual.