Scriptseq v2

Deep sequencing-based identification of the B. subtilis tRNA modifications was performed using RiboMethSeq protocol, allowing us to map 2′-O-methylations by their protection to cleavage [41 (link),42 (link)]. Reverse Transcriptase (RT) misincorporation signatures at RT-arresting nucleotides [43 (link),44 (link)] were extracted from RiboMethSeq data and also from the RT-primer extension, using TTO-based ScriptSeq v2 (Illumina, San Diego, CA, USA) library preparation kit. RiboMethSeq analysis of tRNAs was performed using alkaline fragmentation of total B. subtilis RNA, followed by library preparation and sequencing [42 (link)]. RT misincorporation signatures were extracted from Samtools mpileup format and manually validated by inspection of aligned reads in *.bam file in IGV. Pseudouridine mapping (Psiseq) was performed in the tRNA-enriched fraction of B. sublitis RNAs, using a chemical-based protocol derived from CMCT (1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate)-chemical mapping protocol, and specifically adapted to tRNAs [45 (link),46 (link),47 (link)].

RNA was prepared from purified adult cardiomyocytes as described (He et al., 2012a (link)). mRNA sequencing library was prepared with Script-seq v2 (Illumina), and sequenced on Illumina Hi-seq 2000 (PE100). The resulting sequences were mapped to the mouse genome mm9 with STAR (Dobin et al., 2013 (link)). Transcripts per million reads (TPM) and Fragments per kilobase of exon per million fragments (FPKM) were generated for further quantification by RSEM and Bowtie 2 (Langmead and Salzberg, 2012 (link); Li and Dewey, 2011 (link)), and differentially expressed genes were called with edgeR (v3.14.0) with the following criteria: adjusted p-value<0.05 and fold change (FC) >1.5 or <0.67. The gene ontology (GO) enrichment analysis of DEGs was performed using Metascape (Tripathi et al., 2015 (link)). The top six GO terms with p-value<0.001 in the ‘biological process’ category were used.

MG1655+FosC, MG1655::LPLσ+FosC, MG1655+Fos10T31 and MG1655::LPLσ+Fos10T31 (all in duplicate) were grown in LB broth and induced with 1 mM IPTG for 6 h at which time 10-ml culture was sampled for RNA isolation. RNA isolation samples were centrifuged at 4,000 relative centrifugal force (RCF) at 4 °C for 10 min, decanted and stored at −80 °C. Ten μg per sample of RNA extracted with the RNeasy kit (Qiagen) had DNA removed with the DNA-free kit (Life) and was enriched for mRNA with the MicrobExpress kit (Ambion) thrice, according to the manufacturers' protocols. The ScriptSeq v2 (Illumina) kit was used to construct the RNAseq libraries. The fragment length of the libraries was checked using Bioanalyzer (Agilent, Santa Clara, CA, USA) before sequencing.
Deep sequencing using HiSeq2500 (Illumina) with a 75-bp read length resulted in individual library sequence files. Files were processed to remove barcodes, trim adaptors and obtain read counts. Rockhopper software45 (link) was used to align raw read files to the E. coli and L. plantarum genomes (annotations from National Center for Biotechnology Information) and to do differential expression analysis (at q-value<0.05). Integrated Genome Viewer was used for visualizing read alignments46 (link).

rRNA was depleted with Ribo-Zero Gold rRNA Removal kit (Illumina Inc.) and sequencing libraries were prepared with Nextera (Illumina; ventral hippocampus) or ScriptSeq v2 (Epicentre; medial prefrontal cortex) RNA-seq library preparation kits. Sequencing was conducted on HighSeq 2000 (ventral hippocampus, paired-end 91 bp, Illumina) or NextSeq 500 platforms (medial prefrontal cortex, single-end 96 bp; Illumina). Low abundant genes were filtered, keeping genes with at least 1 CPM in at least six samples. With this threshold, we detected 18,947 genes in the medial prefrontal cortex and 19,560 genes in the ventral hippocampus.