GSM7928363: Ki-U2AF1-WT-1, bulk RNA-seq; Homo sapiens; RNA-Seq
live GFP+ CD34+ cells were sorted by flow cytometry and submitted for sequening For single cell RNA 3' sequencing, Sorted transduced CD34+ cell concentration and viability were evaluated using a Countess II FL Automated Cell Counter (ThermoFisher Scientific). Samples were normalized for input onto the Chromium Single Cell A Chip Kit (catalog number #PN120236, 10x Genomics), and single cells were lysed and barcoded for reverse transcription, as previously described.9 Briefly, libraries were sequenced using the NovaSeq6000 SP 100-cycle flow cell (Illumina). Sequencing analysis was performed using the 10x Genomics' Cell Ranger software.   For single cell 5' sequencing, Sorted CD34+ cells from NIH-39 were processed for 5' scRNA-seq library preparation using Chromium Next GEM Single Cell 5' kit v2 (10x Genomics, catalog number #PN-1000265). Libraries were sequenced on the Illumina NovaSeq 6000 system. Single cell expression measurements (digital counts) were obtained from raw sequence data (FASTQ) with the 10x Genomics Cell Ranger pipeline v4.0.0 (default settings, the GRCh38 reference).  For bulk RNA sequencing, Total RNA from sorted U2AF1S34 or U2AF1wild-type transduced CD34+ cells was purified using the PicoPure RNA Isolation Kit (catalog number #KIT0204, ThermoFisher Scientific). Quality checks and quantifications were performed with the 2100 Expert Bioanalyzer (Agilent). Illumina low input total RNA libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (catalog number #634412; Takara Bio), as previously described.15 Libraries were combined and sequenced on the Illumina NextSeq500 sequencer using the High Output 150 cycle flow cell with the 100nt PE format.