NA24149 RNA-seq
Cells were obtained from Coriell and cultured according to instructions (upright with unfiltered lids overnight then with filtered lids and antibiotics). One million cells from each strain were then flash frozen in lysis buffer (1%BME in RLT). Four replicate RNA extractions were done for each strain using the RNeasy Mini Kit (QIAGEN). RNA-Seq library prep was done with a modified version of the SMART-Seq2 protocol. Whole transcriptome amplification was performed using KAPA HiFi PCR Master mix (Kapa Biosystems KK2602) and IS PCR Primer (IDT). Size selection was done using Ampure DNA SPRI beads at 0.6x and 0.7x concentrations. Concentration was quantified using a Qubit. Sequencing libraries were prepared using the Nextera XT DNA tagmentation kit (Illumina FC-131-1096) with 250 pg input for each sample according to the manufacturers instructions, and Ampure DNA SPRI bead size selection at 0.6x and 0.8x was done. Paired end sequencing was done using Illumina 300 Cycle NextSeq500/550v2 kit with loading density at 2.2 pM.