title: RNA-seq of Nalm6 cells: SF3B1 K700E, Capture, rep2
design: 10 g of total RNA from SF3B1 K700K or SF3B1 K700E knocked-in NALM-6 cell lines were treated with RibominusTM Eukaryotic Kit (Life Technologies, Cat#K15020) to remove the ribosomal RNA. The resulting RNA was homogenized by 0.25 U RNase III (Ambion, Cat#2290)/g RNA/10 ul reaction at 37 °C for 10 min, followed by phenol/chloroform extraction and ethanol precipitation to remove the RNase. Reverse transcribe 250 ng of the homogenized RNA with SuperScript II Reverse Transcriptase (Invitrogen, Cat#18064) to make the cDNA. To generate Illumina-compatible cDNA library, the cDNA is subject to second strand cDNA synthesis using NEBNext® mRNA Second Strand Synthesis Module, end-repair with NEBNext® End Repair Module, dA-tailing with NEBNext® dA-Tailing Module, ligation with Illumina universal and barcoded adaptors with NEBNext® Quick Ligation Module, and finally amplified by NEBNext® High-Fidelity 2X PCR Master Mix and the outer primers (5'AATGATACGGCGACCACCGAGATCTACAC and 5'CAAGCAGAAGACGGCATACGAGAT).  To enrich the cDNA that crosses the 5’ss- branchpoint junction, the cDNA library of SF3B1 K700K and K700E NALM-6 cells are combined and hybridized with biotinylated RNA probes targeting 5’ end of introns.  Alternatively, the cDNA library was treated by duplex-specific nuclease  (DSN, Evrogen, Cat#EA001) to enrich the rare species following the Illumina DSN normalization protocol.  cDNAs across the branchpoint junction was enriched by various rounds of treatments: DSN only, capture only, capture+DSN, capture+capture, capture+DSN+capture, and capture+DSN+capture+DSN. The enriched cDNA library was sequenced using HiSeq paired-end 100 bp protocol at BGI Hong Kong.