title: RNA Seq from HeLa cell line NCL_KD condition set_1
design: RNA isolation was performed from three independent biological replicates of MALAT1+/+, MALAT1-/-, FL, Q123m and NCL KD cells, as per the protocol mentioned earlier in Methods. A modified NEBNext RNA Ultra II directional protocol was used to prepare the libraries for total RNA sequencing. The first step for purifying the poly-A containing mRNA molecules using oligo-dT attached magnetic beads was omitted. Instead, the first step involved the removal of ribosomal RNA of cytoplasmic and mitochondrial origin using biotinylated, target-specific oligos combined with rRNA removal beads. Following purification, the RNA was fragmented using divalent cations under elevated temperature. Next, the cDNA was synthesized using Reverse transcriptase and random hexamers in a first strand synthesis reaction. Subsequently, the cDNA was converted to double stranded cDNA where Uracil was added instead of Thymine. The strand specificity was preserved by a USER enzyme-based digestion of the second strand thereby leaving one functional strand which mapped to the DNA strand which it was transcribed from. The USER digested single strand molecules were enriched and indexed in a limited cycle PCR followed by AMPure bead purification to create a final cDNA library for sequencing. Prepared libraries were sequenced on Illumina Novaseq to generate 80M, 2x150bp reads/sample. Upto 75% of the sequenced bases were of Q30 value. Sequenced data were processed to generate FASTQ files.