GSM7986195: In vitro sample 1 Treated; Bacillus subtilis; RNA-Seq
To extract the RNA from the Flag-IP or from the in vitro reaction 1 ml of Trizol reagent (Invitrogen) was used according to the manufacturer's instructions with an additional 75% EtOH wash step at the end. The samples were then taken into a modified NextFlex small RNA seq v4 protocol. Inputs were standardized to 206 ng as measured by RNA Qubit HS. Samples had 3' adapters ligated with an adapter dilution of 1:1, followed by an adapter inactivation step (Steps A and B). Samples were cleaned up with Adapter Depletion Solution, beads, and isopro-panol following Step E but with reagent volumes adjusted for the smaller reactions. Sam-ples were resuspended in 12 µl water, and 11.2 µl were taken, added with 4 µl of 5X T4PNK buffer (NEB), and fragmented at 94°C for 1 min. 4 µl of 10 mM ATP and 0.8 µl of T4PNK were added, and samples were incubated at 37°C for 30 mins, followed by deac-tivation at 65°C for 20 mins. Then samples were taken into the 5' ligation step from the Nextflex protocol with adapters diluted 1:3 (Step C), and the remainder of the protocol was followed as per manufacturer instructions. The positive control was amplified with 16 PCR cycles, while the DP samples were amplified with 25 PCR cycles. Samples were cleaned up individually with a 1.3x bead ratio, and checked on the bioanalyzer. Samples were pooled equimolarly, and cleaned up once more with a 1x bead ratio. Samples were sequenced using the MiSeq 50 bp v2 kit with the following read mode: 5-8-0-61. Sam-ples were demultiplexed using bcl2fastq, adapter trimming was performed with cutadapt, sequences from Read 2 were reverse-complemented (_rc) and taken forward into alignments.