Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cryopreserved patient samples approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center were thawed and stained using standard procedures (10 min, 4°C) with the surface antibody CD34-PE-Vio770 (clone AC136, Miltenyi Biotec) and DAPI (Sigma-Aldrich). Cells were then sorted for DAPI-negative, CD34+ and CD34-negative cells using BD Influx. For the species mixing experiment, human MPL expressing Ba/F3 and UT-7 cell lines were generated by retroviral transduction, after which they were subjected to infection with CALR variant lentiviral supernatants. Wildtype UT7 cells and mutant Ba/F3 cells were mixed in equal proportions. The standard 10x Genomics Chromium v.2 protocol was carried out according to manufacturer's recommendations (10x Genomics, Pleasanton, CA) until after emulsion breakage and recovery of first strand cDNA. If the targeted gene of interest, e.g. SF3B1, was not robustly detected by the standard 10x procedure (i.e. if <60% of the expected cells showed expression) based on a priori knowledge in a similar dataset, a gene-specific primer was spiked into 10x primer mix at 1% of the concentration of the cDNA amplification primers for the initial cDNA PCR step. Then, during the amplification step, the 10x cDNA library underwent an extra cycle of PCR beyond the manufacturer's recommended number of cycles. After cleanup with SPRIselect, a small portion of the cDNA library, 3 µL (~10% of total) was aliquoted for targeted genotyping, and the remaining cDNA underwent the standard 10x protocol. The cDNA set aside for GoT was amplified for 3 to 4 additional cycles using KAPA HiFi HotStart ReadyMix (KAPABiosystems) and 10x primer mix to provide sufficient material for the enrichment step. After clean-up, locus-specific reverse primers and the generic forward SI-PCR were used to amplify the site of interest of the cDNA template (Supplementary Table 3) using ~10 PCR cycles. The locus-specific reverse primers contain a partial Illumina read 2 handle, a stagger to increase the complexity of the library for optimal sequencing and a gene specific region to allow specific priming. The SI-PCR oligo (10x) anneals to the partial Illumina read 1 sequence at the 3' end of the molecule, preserving the cell barcode (CB) and UMI. After the initial amplification and SPRI purification to remove unincorporated primers, a second PCR was performed with a generic forward PCR primer (P5_generic) to retain the CB and UMI together with an RPI-x primer (Illumina) to complete the P7 end of the library and add a sample index. The targeted amplicon library was subsequently spiked into the remainder of the 10x library to be sequenced together on a HiSeq 2500 or sequenced separately on MiSeq with v3 chemistry (Illumina). The cycle settings were as follows: 26 cycles for read 1, 98 or 130 cycles for read 2, and 8 cycles for sample index.