GSM8465433: gDNA_quant_MPRA_6; Bacillus subtilis; OTHER
RNA was extracted from frozen cell pellets with gDNA depletion using an RNeasy Plus Mini Kit (Qiagen). gDNA was prepared from a frozen cell pellet using the Wizard Genomic DNA Purification Kit (Promega). For RNA-derived libraries from experiments 1a, 1b, 2a, 2b, and 9 (see Table S7), 40 µg of total RNA was reverse transcribed using primer oJT442 and SuperScript III. This cDNA was then run on a 6% TBE-Urea gel, and the following size selection ranges were gel purified: 300-500 nt for full-length cggR and glnR constructs (experiments 1a, 2a, 9) and 100-250 nt for cleaved cggR constructs (experiments 1b and 2b). For the remaining experiments, cleaved RNA was isolated by size selection of RNAs ranging from 200-400 nt for tetM-cggR sites, 150-300 nt for aprE-cggR sites, and 150-350 nt for aprE-glnR sites on a 6% TBE-Urea gel (Novex). The size selected product was reverse transcribed using primer oJT442 and SuperScript III (Thermo Fisher) and size selected on 6% TBE-Urea gel, extracting approximately 120 to 200 nucleotides for cggR libraries and 100 to 200 for glnR. For all experiments, size-selected cDNA was amplified using Phusion DNA polymerase (NEB), adding a barcode and Illumina adapters with a Truseq indexing primer and an isoform-specific primer (see Table S2 for primer sequences). These isoform-specific primers were oJT441/oJT1290 for cleaved/full-length products from cggR constructs or oJT890/oJT1291 for cleaved/full-length products from glnR constructs. gDNA-derived libraries were generated using a two-step Phusion PCR protocol. An initial amplicon was performed 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 the same primers as used for quantifying cleaved RNA abundance (oJT441 or oJT890 with our Truseq indexing primer) and size selected on an 8% TBE gel.