Neb ultra 2 fs kit

Bacterial cryo-cultures of the different isolates were revived on LB agar plates. Single colonies were picked and grown overnight in 2 ml LB medium at 37°C. The cells were pelleted by centrifugation. Cells were lysed and genomic DNA was extracted using a Nucleospin Tissue Kit (Machery and Nagel). Approximately 150–1,000 ng of DNA were used for fragmentation (insert size: 300–700 bp) and sequencing libraries were prepared following the NEB Ultra II FS Kit protocol (New England Biolabs). Libraries were quantified using a JetSeq Library Quantification Lo-ROX Kit (Bioline) and quality-checked by Bioanalyzer (Agilent). These libraries were sequenced on an Illumina MiSeq instrument with a final concentration of 8 pm using the v3 600 cycles chemistry and 5% PhiX.

Library preparation for bulk‐sequencing of poly(A)‐RNA was done as described previously.^[35] Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo‐dT primer containing barcodes, unique molecular identifiers (UMIs), and an adaptor. 5′‐Ends of the cDNAs were extended by a template switch oligo (TSO) and full‐length cDNA was amplified with primers binding to the TSO‐site and the adaptor. NEB UltraII FS kit (New England Biolabs) was used to fragment cDNA. After end repair and A‐tailing a TruSeq adapter was ligated and 3’‐end‐fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al. (2016), the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2. Data were processed using the published Drop‐seq pipeline (v1.0) to generate sample‐ and genewise UMI tables. Principal component analysis and heat map generation was carried out by the R package Pheatmap.