GSM5034504: ME_0h3; Hordeum vulgare; RNA-Seq
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Agilent 2100 with RIN number >7.0. Poly(A) RNA is purified from total RNA(5ug) using poly-T oligo-attached magnetic beads using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA, which were next used to synthesise U-labeled second stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP. An A-base is then added to the blunt ends of each strand, then ligated to modified Illumina multiplex barcode adapters, which including custom Unique Molecular Identifiers for minimizing sequence-dependent bias and amplification noise according to (Shiroguchi et al. 2012), and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, The ligated products are amplified with PCR by the following conditions: initial denaturation at 95C for 3 min; 8cycles of denaturation at 98C for 15 sec, annealing at 60C for 15 sec, and extension at 72C for 30 sec; and then final extension at 72C for 5 min. The average insert size for the final cDNA library was 300 bp (+-50 bp). At last, we performed the paired-end sequencing on an Illumina Novaseq 6000 (LC Bio, China) following the vendor's recommended protocol.
GEO Accession
GSM5034504