3 protocols
Basecalls performed using Illumina CASAVA_v1.8.0 Filter out the reads that did not pass the quality filter by Illumina CASAVA 1.8 Sequenced reads were trimmed for adaptor sequence, then mapped to Shewanella amazonensis SB2B genome using bowtie v0.12.7 with parameters -t -q -v 2 -m 1 Peaks were called by counting the total number of reads whose starts mapped to the genome location Genome_build: Shewanella amazonensis SB2B/ASM1524v1 Supplementary_files_format_and_content: wig file per sample per scaffold per strand
nucleic acid library construction protocol
Bacterial pellets were collected by centrifuging cultures for 10 minutes at 10,000 g at 4 C in RNAse free 50ml polypropylene tubes. Supernatant was immediately poured off and pellets were stored at -80 C. After thawing, RNA was extracted with RNeasy miniprep columns (Qiagen) with the optional on-column DNase treatment. RNA quality was confirmed with an Agilent Bioanalyze. Ribosomal RNA was depleted with the MICROBExpress kit (Ambion), which uses magnetic beads that bind to oligonucleotides that hybridize to ribosomal RNA. We used terminator 5'-P-dependent exonuclease to degrade transcripts with non-triphosphate 5' ends, TAP phosphatase to convert 5'-PPP to 5'-P ends, blocked the 3' ends with sodium periodate and we added a sequencing adaptor onto the 5' end with Ambion T4 RNA ligase. We used random hexamer primers with a sequencing adaptor on their 5' end to obtain first-strand cDNA. We PCR amplified the library to enrich for products that contained both adaptors and to complete the 5' adaptor. 6-mer index GATCAG was incorporated into the reverse PCR primer. We purified the PCR products and removed unincorporated nucleotides, primers, and adaptor-only products with AMPure XP Beads (Agencourt).
Aerobic growth to mid-log phase at 30C with shaking at 200 rpm