Take3 microspot plate reader

3.33 µg of the isolated genomic DNA was used to amplify the bar-coded amplicons in 39 Herculase II DNA polymerase (Agilent cat. 600679, Santa Clara, CA) reactions per sample (primers described in Supplementary Data 13). 5 µl amplicon or 1 µl diluted plasmid library was used as template in 13 50 µl Herculase II DNA polymerase reactions per sample to attach pooled variable-length spacers and Illumina indexes (primers described in Supplementary Data 13). 24 cycles were used to amplify DNA in the first and second PCR, respectively. The amplicon fragments after PCR 2 have the following sequence (354–362 bp library with variable 20 bp sgRNA sequence in the middle) (SF1). DNA was pooled by sample and purified using the Nucleospin Gel and PCR Clean-up kit (Clontech cat. 740609.250, Mountain View, CA). DNA was quantified using a Qubit high-sensitivity DNA quantification assay (Thermo Fisher cat. Q32851) and Take3 microspot plate reader (BioTek). DNA quality was analyzed by Experion CHIP assay (BioRad cat. 7007-163, Hercules, CA). Clusters were generated on the flow cell using the HiSeq Rapid Duo CBot Sample Loading Kit (Illumina CT- cat. 403-2001, San Diego, CA). A single-read rapid run of 75 cycles was performed on a HiSeq. 1500 (Illumina cat. GD-402-4002) using the HiSeq Rapid SBS kit (Illumina cat. FC-402-4022) with 10% PhiX.

After 8 days of transduction a T0 sample was collected (N = 2) and the remaining library-transduced cells were treated with 15 mM APAP for 30 minutes up to 4 days (2 biological replicates for T0, 24 hour, and 4 day samples). Samples that underwent 4 days of APAP treatment were outgrown for 21 days prior to collection. Genomic DNA was isolated from samples of a minimum of 2E7 cells using the Blood and Cell Culture Midi Kit (Qiagen cat. 13343, Valencia, CA), resulting in a minimum of 136 µg DNA per sample. DNA was quantified using the Qubit high-sensitivity DNA quantification assay (Thermo Fisher cat. Q32851) and Take3 microspot plate reader (BioTek Epoch, Winooski, VT).

Genomic DNA (gDNA) was extracted from puromycin selected cells using the Gentra Puregene Cell Kit (Catalog#: 51306, QIAGEN, Toronto, Canada) according to the manufacturer’s instruction. 50 ng of the isolated genomic DNA was used as template to amplify DNA by Platinum Taq (Catalog#: 10966-026, Thermo Fisher, Waltham, MA, USA) PCR. The amplicon DNA after PCR was verified by 2% agarose gel and gel purified using the Nucleospin Gel and PCR Clean-up kit (Catalog#: 740609.250, Clontech, Mountain View, CA, USA). The concentration and purity of DNA was determined by measuring absorbance at 260 and 280 nm using Take3 microspot plate reader (BioTek), and the nucleotide sequence of individual colonies was determined by sequencing using the following primer: Human SLC26A3_R: 5′-TCCCAAAGTGCTGGGATTAC-3′ (Lot: 162860013); Mouse Slc26a3_R: 5′-TACTGATGCAGCCACCATTAC-3′ (Lot: 190941198).