GSM8553251: Heat at 49C, biol rep 2; Escherichia coli; RNA-Seq
The extraction protocol focuses on isolating RNA from the resuspended inclusion body pellet. Begin by preparing the RNA extraction setup using the Aurum Total RNA Mini Kit from Bio-Rad, following the manufacturer's instructions. Resuspend the final pellet in an appropriate volume of the lysis buffer provided in the kit, then add lysozyme and proteinase K to the mixture, which aids in breaking down cellular components. Allow the mixture to incubate at room temperature for 10 to 30 minutes, facilitating the lysis of aggregated proteins and cellular debris. After the incubation, proceed with the RNA purification steps outlined in the Aurum Total RNA Mini Kit. This usually involves mixing the lysate with a binding buffer and transferring it to a spin column designed to capture RNA while washing away impurities with a wash buffer. Finally, elute the purified RNA using RNase-free water, ensuring that all RNA is collected effectively. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using the Bioanalyzer 2100 (Agilent).  For each sample, 100 ng of total RNA was used as input, using the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus Kit (protocol version: #1000000124514 v02 (April 2021)). Initially, abundant rRNA molecules are first depleted from total RNA by means of DNA probes which bind to the rRNA molecules; both are then enzymatically digested. RNA-specific magnetic beads were used to recover and purify the rRNA-depleted total RNA. The purified total RNA was fragmented and in a reverse transcription reaction using random primers and actinomycin D. RNA was converted into single-stranded cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using dUTP to achieve strand specificity. The cDNA fragments were extended with a single adenosine base to the 3' ends of the blunt-ended cDNA fragments after which pre-index anchors were ligated preparing the fragments for dual indexing. Anchor-ligated fragments were then purified using magnetic beads. Finally, enrichment PCR was carried out to enrich those DNA fragments that have anchor-ligated DNA fragments and to add indexes and primer sequences for cluster generation.
GEO
GSM8553251_r1