Rna hs assay kit

For RNA-seq experiments, virgin flies were collected, and 50 antennae were handpicked, either immediately or after 4 or 14 days on standard Drosophila culture medium at 24°C. Total RNA was extracted using TRIzol (Invitrogen, cat. no. 15596–018) according to the manufacturer’s instructions. DNA was degraded using the Invitrogen TURBO DNA-free Kit. After DNase treatment, TRIzol RNA extraction was repeated a second time. The concentration and quality of the RNA was determined using a sensitive fluorescent-dye-based Qubit RNA HS Assay Kit and the Agilent HS RNA kit and an Agilent 4200 TapeStation System.
Using 1–5 μg of total RNA for each sample, we performed 2 rounds of mRNA isolation using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) according to the manufacturer’s instructions. Libraries were generated using the NEBNext RNA Ultra II RNA Library Prep Kit. The samples were quality controlled and successfully sequenced on an Illumina NextSeq 500 next-generation sequencing system in mid-output mode via 1 × 100 bp paired-end sequencing.

10 ml of B. subtilis at OD 0.1–0.2 was centrifuged in 15‐ml falcon tubes for 10 min at 3,000 g (4,000 rpm). Cell pellets were resuspended in 1 ml of Qiagen RNAprotect Bacteria Reagent and flash frozen in liquid nitrogen.
Defrosted cells were pelleted by a quick centrifugation. After removing the supernatant, cells were resuspended in 1 ml buffer RLT from Qiagen RNeasy kit. Resuspended cells were transferred in a Fastprep Lysing Matrix B tube (MP Bio) and processed in Fastprep apparatus 45 s at speed 6.5 M/s. 700 µl of the supernatant, containing lysed cells, was transferred to a new microcentrifuge tube, to which 500 µl of absolute ethanol was added. After vortexing, the lysate was transferred to a RNeasy spin column and centrifuged 15 s at > 9,400 g (10,000 rpm). RNA purification was then carried out following the instructions of the Qiagen RNeasy kit. RNA quality and integrity were assessed on the Agilent 2200 TapeStation, and RNA concentration was assessed using Qubit RNA HS assay kit. Library preparation was performed using ScriptSeq™ Complete Kit (Illumina, BB1224), for 2 µg of high integrity total RNA (RIN > 8). The libraries were sequenced on a NextSeq500 using paired‐end sequencing of 75 bp in length.