WT1_R1_001
The RNA from frozen lysates extracted using the Qiagen RNAeasy extraction kit as per the standard kit protocol. Subset of extracted RNA samples are analysed for quality by Agilent RNA Tapestation and RIN scores obtained. RIN values were >6.5. For RNAseq, the samples were DNAse treated and ribosomal RNA depletion was performed on all samples. All the rRNA depleted RNA samples were taken through for cDNA conversion with OligodT primers using an adapted Smartseq assay (32). Briefly, the first strand of the cDNA is synthesized with the addition of a few untemplated C nucleotides using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). The poly(C) overhang is added exclusively to full-length transcripts. An oligonucleotide primer (TSO - Transfer Switching Oligo) is hybridized to the poly(C) overhang and used to synthesize the second strand. The Full-length cDNAs were then amplified using KAPA HiFi DNA Polymerase (Roche Diagnostics), purified, and quantified using Promega Quantus Fluorometer. The double-stranded cDNA was further prepared for sequencing using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs) and sequenced on the Novaseq 6000 using 150bp PE Chemistry (Illumina Inc).