mKir2.1 indel deep mutational scanning, FACS sorted pool, gate 3, replicate 3
Genomic DNA was extracted from sorted cells using a Micro kit from Zymo. Following DNA extraction and quantification with NanoDrop, 1.5 g of each library was used as template for PCR using specific primers with PrimeSTAR GXL enzyme, with a final primer concentration of 0.25 M each, and a Tm of 56C and 18 cycles. The amplified bands were then run on a 1.5% gel and extracted. The eluted bands were quantified using Qubit with HS kit. Amplicons were prepared for sequencing using the Nextera XT DNA Library kit from Illumina with 1 ng of DNA input. Samples were indexed using the IDT for Illumina UD indexes and SPRISelect beads at a 0.9x ratio were used for cleanup and final size selection. Each indexed tagmented library was quantified with Qubit HS as well as Agilent 4200 TapeStation. Samples were then pooled and sequenced on a NovaSeq 6000 SP300 flowcell in paired-end mode.