title: GSM6001956: Bacillus coagulans control [EA1]; Weizmannia coagulans; RNA-Seq
library_construction_protocol: Both the samples of control and treatment were stabilized by RNAprotect Bacteria Reagent 2×100 ml. Two tubes of required volume of bacterial culture and double volume of RNAprotect Bacteria Reagent were mixed by vortexing for few seconds (individual tubes for treatment and control). Then the mixtures were incubated for 5 min at room temperature (15-25˚C) and centrifuged for few min at ×g. After that, the supernatants were discarded;the pellets were kept in a freezer. The RNA was expected to remain intact in a deep freezer for up to one month. On-column enzyme digestion was performed to digest and remove genomic DNA. Concentrations of RNA were measured with the NanoDrop, while RNA quality was assessed. Samples included in the present analysis had a RIN score greater than 6. For library preparation, “Microbe express kit” was used for mRNA capture by oligo mag beads, followed by mRNA fragmentation. Next, the cDNA is synthesized using Reverse transcriptase and random hexamers in a first strand synthesis reaction. Subsequently, the cDNA is converted to double stranded cDNA where Uracil is added instead of Thymine and adds dAMP to the 3′ ends of the resulting dscDNA. In Adapter ligation, where dsDNA adapters with 3′ dTMP overhangs are ligated to library insert fragments followed by library amplification, to amplify library fragments carrying appropriate adapter sequences at both ends using high-fidelity, low-bias PCR.