GSM7781566: sorted bacteria, 16S-high, rep3; Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S; OTHER
Genomic DNA was extracted with DNeasy Blood & Tissue, following the instructions for gram-negative bacteria. DNA was extracted from 5 million sorted bacteria using DNeasy Blood & Tissue (Qiagen, 69504) following manufacturer's instructions, with an overnight proteinase K digestion step (which also serves as PFA de-crosslinker), and resuspension in 200 µl of resuspension buffer. DNA (∼ 20 ng per sample) was processed as previously described (Schwartzman et al., Nature Methods 2016), with few modifications. Briefly, DNA was sonicated at 4◦C (program: 6 cycles of 30 s on, 60 s off) on a Bioruptor Plus (Diagenode), achieving a size distribution between 200-1000 nucleotides (nt), with 450 nt average size. The sonicated DNA was end-repaired for 30 minutes at 20◦C in 200 µl final volume containing 1x end-repair buffer and 10 µl of end-repair mix (NEB, E6050L). The reaction was cleaned in 2x Ampure XP Beads (Beckman Coulter, A63881) and eluted in 37.5 µl. A-tailing was performed 20 min at 37◦C in 50 µl final volume, containing 1x NEBuffer 2 (NEB, B7002S), 1 mM ATP, and 125U of Klenow fragment (NEB, M0212M). The reaction was cleaned in 2x Ampure XP Beads (Beckman Coulter, A63881) and eluted in 44 µl. DNA was then dephosphorylated overnight at 37◦C in 50 µl final volume, containing 1x rCutSmart buffer (NEB, B6004S), and 5U of quick CIP (NEB, M0525L). The day after, the reaction was heat-inactivated for 2 minutes at 80◦C, cleaned in 2x Ampure XP Beads (Beckman Coulter, A63881), and eluted in 18 µl. The DNA template was ligated for 20 min at 25C in 50 µl final volume, containing 0.22 µM Illumina-compatible forked indexed adapters, 1x buffer, and 5 µl of quick ligase (NEB M2200L). To release the non-ligated strand of the adaptor, DNA was denatured at 96◦C for 5 min, cleaned with 0.8x Ampure XP beads (Beckman Coulter, A63881), and eluted in 30 µl. Two PCR reactions were used for TIS library construction. The first PCR reaction was performed in triplicate in 25 µl final volume containing 0.5 µM TN bait primer 1, 0.5 µM Illumina enrichment primer, 1x KAPA HiFi HotStart ReadyMix (Roche, KK2601) and 10 µl DNA template. PCR program: 60s 98◦C, 15 cycles of 15s 98◦C, 20s 68◦C, 60s 72◦C, and final extension of 60s 72◦C. Following the first PCR reaction, the amplified DNA from the three reactions was pooled and cleaned with 1x Ampure XP beads (Beckman Coulter, A63881), and the eluate was used as a template in the second PCR reaction. Conditions for the second PCR reaction were identical to the first, with the following differences: reaction was performed in a single 50 µl reaction and TN bait primer mix 2 consisted in a mix of staggered nested primers. Libraries were quantified with NEBNext Library Quant Kit for Illumina (NEB, E7630L) and sequenced on a Novaseq machine with the following settings: R1=85, I1=8, and R2=37.