Direct zol miniprep columns

TRIzol reagent (Thermo Fisher) was added to 100 μl of the C-P7 stock or 200 μl of the A-P2 stock, and RNA was extracted using Direct-zol miniprep columns (Zymo Research). Reverse transcription was performed using oligo(dT) with Superscript IV (Thermo Fisher) at a RT temperature of 60°C for 10 min. The cDNA was then used in PCRs with PfuUltra II Fusion HS DNA polymerase (Agilent) and with previously published primer combinations for sequencing of the VA1 genome (31 (link)). For sequencing of the VA1 isolate from A-P2, two changes were made to the PCR protocol. First, the number of PCR cycles was increased to 40 for all primer combinations of the genome. Second, the annealing temperature for fragments 1 and 4 was increased to 58°C. The genome from each stock was sequenced in three independent experiments, and genetic variants were identified if they were present in at least two of the three experiments.

2e7 K562 cells per replicate were pelleted at 500×g for 5 min at 4 °C and then resuspended in 1× PBS buffer. Two replicates were performed for each sgRNA. RNA extraction was performed as follows: 500 µL of TRIzol was added to each sample, mixed by inverting the tube, and then 5 min later 100 µL of chloroform was added. Samples were spun at 12,000 × g for 15 min at 4 °C. The aqueous layer was transferred to an RNase-free tube and mixed with 300 µL of 70% ethanol and vortexed. Contents were then transferred to Direct-zol Miniprep columns (Zymo) and the protocol was followed according to the manufacturer’s instructions, including the on-column DNaseI treatment. RNA was eluted in 15 µL of RNase-free water and stored at −80 °C and a separate 2 µL aliquot was set aside for testing RNA concentration and quality via Nanodrop. RNA-seq libraries were prepared from 700 ng of total RNA with the TruSeq RNA Library Prep kit v2 (Illumina) low sample protocol, which uses oligo-dT beads to enrich for A-tailed mRNAs. Library concentration and length was determined with a 2200 Tapestation System (Agilent) and Qubit (Thermo Fisher Scientific). Libraries were pooled and sequenced on a Nextseq (Illumina).