GSM8367123: WCS23_S_3; Escherichia coli W; RNA-Seq
Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions. RNA concentration was quantified using a NanoDrop OneC spectrophotometer (Thermo Scientific). The quality of the isolated RNA was checked by running 1% agarose gel electrophoresis. One microgram of the RNA from each sample was used for rRNA depletion with riboPOOL (siTOOLs Biotech) and Dynabeads MyOne Streptavidin C1 (Invitrogen), followed by purification using RNA Clean & Concentrator Kit (Zymo Research) according to the manufacturer's instructions. The suitable RNA samples were then used to construct paired-end, strand-specific RNA-seq libraries using KAPA RNA Hyper Prep kit (Kapa Biosystems) with the modification of shortened fragmentation of 2 minutes at 85℃ to include sRNAs in the library. The concentration of the final library was measured with Qubit 4 fluorometer (Thermo Scientific) and the size distribution was checked with Agilent 2100 Bioanalyzer (Agilent). The library was sequenced on NextSeq 550 (Illumina) platform with NextSeq 500/550 High Output Kit v2.5 (Illumina) in accordance with the manufacturer's instructions. All RNA-seq experiments were performed in biological triplicate.