GSM4592706: Col-0_0.5h_avrRpt2_Rep1; Arabidopsis thaliana; RNA-Seq
RNA was purified from plant tissue using the RNeasy 96 Kit (Qiagen). The plant “user developed protocol” was followed except for the addition of a 96% ethanol wash step after the second RPE wash to ensure removal of residual salts. Purified RNAs were kept at -80C. Illumina-based mRNA-Seq libraries were prepared from 1 μg RNA following [61]. Briefly, mRNAs were selected using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences). RNAs were washed and fragmented at 94°C for 6 minutes. First-strand cDNA synthesis was performed using random hexamers and reverse transcriptase (Superscript III reverse transcriptase, Invitrogen). Second-strand cDNA synthesis was done using DNA Polymerase I and RNAseH. Double-stranded cDNAs were end-repaired using T4 DNA polymerase, T4 polynucleotide kinase, and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina adapters (Kapa Dual-indexed adapter kit, Roche). Unless specified, reagents were purchased from Enzymatics. Library quality control and quantification were performed using the 5200 Fragment Analyser and the NGS fragment kit (Agilent). Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.
GEO
GSM4592706