title: GSM8881143: Zea mays shoot apex from homozygous Normal siblings from Trf segregating population in W22 background, replicate 1; Zea mays; RNA-Seq
library_construction_protocol: A W22-backcrossed segregating population of 3-week old maize seedlings were first genotyped for herterozygous Trf versus homozygous normal (N). The outer 2 leaves were removed and 0.5cm of the shoot apex was dissected and flash frozen in liquid nitrogen. Three replicate pools were created for Trf and N siblings, 10 individuals per pool. RNA for all experiments was extracted from each pool using TRIzol. (Invitrogen) as described in the manual. RNA was DNAse treated to remove gDNA contamination using Invitrogen DNaseI (#18068015) digestion. RNA quality was checked using gel electrophoresis (1x Agarose in 1x TBE) and quantified using Qubit before sending for library preparation and 150bp PE sequencing by Novogene. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.