GSM4718506: A.thaliana_CM_rep2; Arabidopsis thaliana; RNA-Seq
Cells (1 ml frozen aliquots) were pulverised into a powder by bead beating at 30 h/z for 1 min. Total RNA was then purified using an RNeasy® plant mini kit (Qiagen™; ref. 74904) according to manufacturer's instructions Library preparation and RNA-seq was performed by Novogene (Beijing, China). A total of 1 μg RNA was used as input material for library generation using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB USA), at which point index codes were added to each sample. mRNA was purified using magnetic beads harbouring poly-dT oligos. Fragmentation was performed using divalent cations in heated NEBNext First StrandSynthesis Reaction Buffer. First strand cDNA was synthesised using random hexamers and M-MuL MuLV Reverse Transcriptase. Second strand cDNA was synthesised using DNA Polymerase I (+RNase H). Blunt ends were generated via exonuclease/ polymerase activities. Next, 3' DNA ends were adenylated before ligating an NEBNext hairpin loop adaptor for hybridization. Fragments of ca. 200 bp were preferentially selected for using an AMPure XP system (Beckman Coulter, Beverly, USA). Purified, adaptor-ligated fragments were treated with 3 µl of USER® Enzyme (NEB, USA) before selective PCR enrichment using Phusion® High-Fidelity DNA polymerase using Universal and index primers. Products were purified again using the AMPure XP system and library quality assessed using the 2100 BioAnalyzer® system.
GEO
GSM4718506