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RNAseq

SK Sivan Kijner
DE Dena Ennis
SS Shimrit Shmorak
AF Anat Florentin
MY Moran Yassour
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Pure cultures of B. dorei growing on various carbon sources (in replicates) were harvested at log phase (OD600 ~ 0.7) using the Direct-zol™ RNA Miniprep Plus kit (Zymo Research, R2071). Agilent 2100 Bioanalyzer (Agilent Technologies) was employed for RNA quality control. Samples were processed according to a previously described protocol89 and sequenced in two separate pools: HMOs (2’-FL, DFL, 3’-SL, 6’-SL, LNT, LNnT) and glucose. Single-end 75 bp sequencing was performed on a NextSeq device and the data was deposited in the National Center for Biotechnology Information (NCBI) repository, accession numbers SRS11934615 (HMOs) and SRS11934616 (glucose)71.

The raw sequencing data were further filtered by trim_galore (https://github.com/FelixKrueger/TrimGalore) and classification of the B. dorei isolate to the strain level was achieved by alignment of reads using BLAST90. Genome mapping to B. dorei DSM 17855 genome (GenBank: CP046176.1) was performed by Bowtie291 and differential expression analysis between HMOs and glucose was carried out using featureCounts92 and DEseq293. DEseq2 uses the Wald test for differential expression analysis and the Benjamini and Hochberg method for multiple hypothesis correction. Significantly up-regulated genes were selected based on the p_adjusted <0.05 and log2 fold-change >1 parameters. Gene annotation of glycoside hydrolases (GHs) was downloaded from the CAZy (Carbohydrate Active enZYmes)53 database.

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