Hs ngs fragment kit

Cells were collected at 0 h, 24 h and 48 h post-induction as bulk samples, and on days 3, 4, and 6, cells were sorted into Venus-positive and Venus-negative cells using FACSAria III (BD bioscience). Cells were washed with PBS and processed using the Qiagen RNeasy mini kit. RNA was additionally treated with a TURBO DNA-free™ kit (Invitrogen) according to the manufacturer’s protocols. The integrity of RNA was measured using Fragment Analyzer™ (Advanced Analytical). RNAseq-Libraries were prepared using QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen GmbH). LUTHOR 3’-scRNAseq (Lexogen GmbH) libraries were prepared using the manufacturer’s protocol after sorting cells into 5 μl lysis agent. Concentrations and distributions of the libraries were checked with the Fragment Analyzer™ using HS NGS Fragment Kit (Agilent; DNF-474-0500). Libraries were then pooled and sequenced using Illumina Nextseq550 in a single read 75 cycles run.

The quantity and quality of the purified total RNA was assessed using a Thermo Fisher Scientific Qubit 3.0 fluorometer with the Qubit RNA BR Assay Kit (Cat # Q10211, Thermo Fisher Scientific) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Cat # DNF-471, Agilent Technologies, Santa Clara, USA), respectively. Sequencing libraries were constructed using an Illumina TruSeq Stranded mRNA Library Prep kit (20020595, Illumina, San Diego, USA) in combination with TruSeq RNA UD Indexes (Cat # 20022371, Illumina) according to Illumina’s guidelines. The cDNA libraries were evaluated using a Thermo Fisher Scientific Qubit 3.0 fluorometer with the Qubit dsDNA HS Assay Kit (Cat # Q32854, Thermo Fisher Scientific) and an Agilent Fragment Analyzer (Agilent) with an HS NGS Fragment Kit (Cat # DNF-474, Agilent), respectively. Pooled cDNA libraries were sequenced 100 bp single-end on one lane of an Illumina HiSeq 3000 instrument (Illumina). All base call files were demultiplexed and converted into FASTQ files using Illumina bcl2fastq conversion software. The quality control assessments, generation of libraries, and sequencing were conducted by the Next Generation Sequencing Platform, University of Bern.

All isolates underwent short-read sequencing. DNA libraries were prepared from extracted gDNA using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (Illumina, San Diego, CA, USA). The DNA was purified and size selected using Ampure XP beads (Beckman Coulter, Brea, CA, USA) and quantified using the Quant-it PicoGreen dsDNA assay (Thermo Fisher, Waltham, MA, USA). The quality and size distribution of the DNA were assessed on a fragment analyzer using the HS NGS Fragment Kit (Agilent, Santa Clara, CA, USA). The pooled samples were then sequenced on a NovaSeq6000 (Illumina, San Diego, CA, USA) as PE250.
All strains also underwent long-read DNA sequencing. Extracted gDNA was treated with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB). Then, barcodes from the Native Barcoding Expansion 1–12 and 13–24 from Oxford Nanopore Technologies (Oxford Nanopore Technologies, Oxford, UK) were ligated using the NEBNext Ultra II Ligation Module (NEB). The DNA was purified using Ampure XP beads (Beckman Coulter). The DNA from different barcoded samples was pooled and the adapter AMII (ONT) was ligated using the NEBNext Ultra II Ligation Module (NEB). Sequencing was performed with an R10.4 MinION Flow Cell using a MinION Mk1B (ONT). All the sequences were deposited in the NCBI under project number PRJNA979211.

Total RNA from PAXgene preserved blood was extracted using the Agencourt RNAdvance Blood Kit (Beckman Coulter, Indianapolis, IN) on a BioMek FX^P Laboratory Automation Workstation (Beckman Coulter). Concentration and integrity (RIN) of isolated RNA were determined using the Quant‐iT™ RiboGreen™ RNA Assay Kit (Thermo Fisher) and an RNA Standard Sensitivity Kit (DNF‐471, Agilent Technologies, Santa Clara, CA, USA) on a Fragment Analyzer Automated CE system (Agilent Technologies), respectively. Subsequently, cDNA libraries were constructed from total RNA using the Universal Plus mRNA‐Seq kit (Tecan Genomics, San Carlos, CA, USA) in a Biomek i7 Automated Workstation (Beckman Coulter). Briefly, mRNA was isolated from purified 300 ng total RNA using oligo‐dT beads and used to synthesize cDNA following the manufacturer's instructions. The transcripts for ribosomal RNA (rRNA) and globin were further depleted using the AnyDeplete kit (Tecan Genomics) prior to the amplification of libraries. Library concentration was assessed fluorometrically using the Qubit dsDNA HS Kit (Thermo Fisher), and quality was assessed with the HS NGS Fragment Kit (1–6,000 bp; DNF‐474, Agilent Technologies).

After SPRI bead purification of the crosslink-reversed samples, we transferred DNA from each to Covaris microTUBE AFA Fiber Snap-Cap tubes (Covaris catalog no. 520045) and sonicated to an average length of 400 ± 85 base pairs using a Covaris ME220 Focused-Ultrasonicator. Temperature was held stably at 6 °C and treatment lasted 65 s per sample with a peak power of 50 W, 10% duty factor and 200 cycles per burst. The average fragment length and distribution of sheared DNA was determined by capillary electrophoresis using an Agilent FragmentAnalyzer 5200 and HS NGS Fragment Kit (Agilent catalog no. DNF-474–0500). We ran sheared DNA samples twice through the NEBNext Ultra II DNA Library Prep Kit for Illumina (catalog no. E7645S) End Preparation and Adapter Ligation steps with custom Y adapters to produce library preparation replicates. We purified ligation products via SPRI beads before Biotin enrichment using Dynabeads MyOne Streptavidin C1 beads (ThermoFisher catalog no. 65002).
We performed indexing PCR on streptavidin beads using KAPA HiFi HotStart ReadyMix (catalog no. KK2602) and PCR products were isolated by SPRI bead purification. We quantified the libraries by Qubit 4 fluorometer and FragmentAnalyzer 5200 HS NGS Fragment Kit (Agilent catalog no. DNF-474-0500) before pooling for sequencing on an Illumina HiSeq X at Fulgent Genetics.