NextSeq 500 paired end sequencing; RNA-seq of coding RNA from USP14 knockout human haploid (HAP1) cells
RNA-seq of coding RNA from USP14 knockout human haploid (HAP1) cells
HAP1 cells were derived from the near-haploid chronic myeloid leukaemia cell line KBM7 as described (Carette et al., 2011). HAP1 cells were cultured in IMDM supplemented with 10% FBS, penicillin-streptomycin and L-glutamine. Cells were incubated at 37°C in 5% CO2 and grown to 80-90% confluence before harvesting total RNA Total RNA from 5 million cells was extracted using Qiagen RNeasy plus mini kit (74134). 1 μg of total RNA was subjected to library synthesis using the TruSeq RNA-Seq kit in technical replicate (e.g. two libraries were made from each RNA sample). 1 μg of total RNA was enriched for Poly-A mRNA using oligo dT-selection and reverse transcribed to cDNA. The cDNA was ligated with Illumina indexes. RNA libraries were prepared for sequencing using standard Illumina protocols.