GSM8465417: BC_mapping_MPRA_9; Bacillus subtilis; OTHER
gDNA was prepared from a frozen cell pellet using the Wizard Genomic DNA Purification Kit (Promega). gDNA-derived libraries were generated using a two-step Phusion PCR protocol. An initial amplicon was generated through 2 cycles of PCR using from 4 µg of purified nucleic acid per 50 µL PCR reaction (reaction scaled up as needed to minimize UMI collapse in more complex libraries) with primers oJT444/445 for cggR upstream mutations, oJT1125/445 for cggR downstream mutations, and oJT891/445 for glnR downstream mutations. The untranslated cggR library required a longer amplicon to capture the upstream stop codon and was instead amplified with oJT942 and oJT445. This first PCR reaction was cleaned up with a double-sided Select-a-Size DNA Clean & Concentrator column (Zymo) and a DNA Clean & Concentrator 5 column (Zymo). A quarter of this reaction was then used for the second round of PCR, using primers oJT446/447. This product was size selected on an 8% TBE gel and sequenced.