HL60 (Acute promyelocytic leukemia cell line)
RNA sequencing libraries were prepared with Illumina-compatible NEBNext  UltraTM Directional. RNA Library Prep Kit (New England BioLabs, MA, USA) at the Genotypic Technology Pvt. Ltd., Bangalore, India.Briefly, 200ng of Total RNA was taken for mRNA isolation, fragmentation and priming. The fragmented and primed mRNA was further subjected to first strand synthesis in the presence of Actinomycin D (Gibco, life technologies, CA, USA) followed by second strand synthesis. The double stranded cDNA was purified using HighPrep PCR magnetic beads (Magbio Genomics Inc, USA). The purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext  UltraTM Directional RNA Library Prep Kit protocol. The adapter ligated cDNA was purified using HighPrep beads and was subjected to 15 cycles of Indexing-PCR (37   C for 15mins followed by denaturation at 98  C for 30 sec, cycling (98  C for 10sec, 65  C for 75sec) and 65  C for 5mins) to enrich the adapter-ligated fragments. The final PCR product (sequencing library) was purified with HighPrep beads, followed by library quality control check. The Illumina-compatible sequencing library was initially quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent Bioanalyzer. Finally, the sequencing library was accurately quantified by quantitative PCR using Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA). The qPCR quantified libraries were pooled in equimolar amounts to create a final multiplexed library pool for sequencing on an Illumina sequencer for 150bp paired-end chemistry.
SUB3400327
WT rep 1