Nextera xt dna library preparation kit 96 samples

A total of 30 isolates were selected for whole genome sequencing based on results from the PCR and MALDI-TOF species identification. Only isolates confirmed as A. hydrophila from the MALDI-TOF analysis were chosen. Sampling year and location were considered so that isolates from all sampling years and provinces were represented. Isolates were cultured on blood agar and incubated at 37°C for 24 h. A pure bacterial colony was transferred to Luria broth (Oxoid) and incubated at 37°C for 13 h. Genomic DNA was extracted using the bacterial DNA extraction kit Maxwell^® RSC Cultured Cells DNA Kit (Promega Corporation, Madison, WI, United States). Quality parameters of the extracted DNA was assessed using NanoDrop™ One (Thermo Fisher Scientific, Waltham, MA, United States) and the DNA concentration using Qubit 2.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States). Whole genome sequencing was performed with Illumina MiSeq (Illumina Inc., San Diego, CA, United States) at Statens Serum Institut (Copenhagen, Denmark) using the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina Inc., San Diego, CA, United States), generating minimum sequence coverage of 50X.

Pools of 3 × 10³ SSCs or OPCs were sorted from the bone fraction into RLT buffer (Qiagen) with 1% of β-mercaptoethanol. RNA was isolated using RNeasy Micro Kit. cDNAs were generated from 400 to 1000 pg of total RNA using Clontech SMART-Seq v4 Ultra Low Input RNA kit for Sequencing (Takara Bio Europe) and amplified with 12 cycles of PCR by Seq-Amp polymerase. For Tn5 transposon tagmentation, 600 pg of pre-amplified cDNAs were used by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina) followed by library amplification of 12 cycles. Purification was performed with Agencourt AMPure XP and SPRIselect beads (Beckman-Coulter). Sequencing reads were generated, in Paired-End mode, on the GenomEast platform (Illumina). FastQC program was used to evaluate the quality of the raw sequencing data and reads shorter than 50 bp were removed. Reads were aligned to the Mus musculus genome (mm10 build) using the Star tool^{90 (link)}. Gene expression quantification was obtained using read counting software Htseq^{91 (link)}. Normalization and differential analysis were carried out with DESeq2 package by applying the Benjamini–Hochberg FDR correction (p < 0.05; 1.5-fold) for comparison between samples. Heatmaps and volcano plots were obtained using the web server Heatmapper and EnhancedVolcano packages, respectively.