Heinz 1706 WT replicate 2
RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, USA) with unique barcodes for each sample. mRNA was isolated using poly-T oligo magnetic beads and fragmented. First-strand cDNA was synthesized with random hexamer primers and M-MuLV Reverse Transcriptase, followed by second-strand synthesis using DNA Polymerase I and RNase H. Blunt-ended cDNA was ligated with NEBNext adaptors, and fragments (150200 bp) were purified using the AMPure XP system (Beckman Coulter, USA). Libraries were amplified via PCR with Phusion High-Fidelity DNA Polymerase (NEB) and assessed for quality on an Agilent Bioanalyzer 2100. Clustering was performed on a cBot system using the PE Cluster Kit cBot-HS (Illumina). Nine cDNA libraries, three per genotype, were sequenced.