GSM7848575: FigS6D: endo dcas9, ΔCRISPR(R24-S2), Cas1/2 + 0.1mM IPTG; Neisseria meningitidis; OTHER
To detect spacer acquisition, 10 ng genomic DNA was used as template to PCR amplify CRISPR's downstream end using Q5 DNA Polymerase with with primers described in Supplemental Table 1. The following PCR conditions were used: 98 ̊C for 30 s; 98 ̊C for 10 s, 60 ̊C for 10 s, 72 ̊C for 20 s (25 cycles); 72 ̊C for 2 min. PCR products were resolved on 2% agarose gel, stained by SYBR Safe (Invitrogen, S33102), and visualized using a Bio-Rad Chemidoc MP imager (12003154). The adapted “+1” “+2” bands are 66- and 132- nts larger than the un-adapted “0” product derived from parental CRISPR array, due to addition of repeat-spacer units (66-nt each). Adaptation efficiency was measured as the percentage of total CRISPR amplicons with new spacers. Quantification of PCR band intensity was done using Image Lab 6.0 software (Bio-Rad). UMI library construction: Ligated products from above were used as template for 1st step Q5 PCR to amplify the leader-end of CRISPR array with primers OYZ3624+593 containing partial adaptor. Cycling conditions are 98 ̊C for 30 s; 98 ̊C for 10 s, 60 ̊C for 10 s, 72 ̊C for 25 s (25 cycles); 72 ̊C for 2 min. Following 2% agarose gel electrophoresis, the “+1” adapted band was gel extracted with QIAquick Gel Extraction Kit (Qiagen, 28706). Eluate is used as template for 2nd step Q5 PCR amplification using I5 and I7 index primers to add full Illumina adapter and indexes. 2nd PCR conditions are: 98 ̊C for 30 s; 98 ̊C for 10 s, 65 ̊C for 10 s, 72 ̊C for 25 s (15 cycles); 72 ̊C for 2 min.   Non-UMI library construction: For mutant strains carrying the Δcrispr::R26, Δcrispr::crispr(sp1), Δcrispr::crispr(sp1-2), Δcrispr::crispr(sp1-3), Δcrispr::crispr(sp1-5), Δcrispr::crispr(sp1-7), Δcrispr::crispr(sp1-8), Δcrispr::crispr(sp1-17), Δcrispr::crispr(sp1-25), Δcrispr::crispr(sp1-49) alleles, a non-UMI method was used due to the lack of unique restriction site (e.g., FatI) close enough to the newly acquired spacer. 10 ng of total genomic DNA from an adaptation experiment was used as template for 1st step PCR with primers are described in Supplemental Table 1. The following steps for “+1” adapted band gel extraction and 2nd step Q5 PCR were as described in the earlier paragraph.  All final libraries from 2nd step PCR were pooled together, cleaned up using QIAquick PCR Purification Kit, and then purified as one sample by agarose gel electrophoresis and gel extraction. This final pooled library was sequenced on an Illumina sequencing platform, NovaSeq (2x150 bp) at the Univ. of Michigan Advanced Genomics Core.