GSM8146729: glp-1(e2144)_atfs-1 RNAi_rep1; Caenorhabditis elegans; RNA-Seq
800 adult animals were collected for each replicate and transferred into 1 ml of M9 Buffer in a 1.5 mL microcentrifuge tube. Worms were pelleted at 500xg for 1 minute, supernatant removed and then washed once with 1 ml of fresh M9, and then once with 1 ml of M9 supplemented with 0.01% Tween-20 following the same spin and supernatant removal. After removing as much of the last supernatant as possible, 250 ul of Qiazol was added to each sample and snap-frozen in liquid nitrogen. Samples were then run through 3 freeze thaw cycles: 37°C bead bath for 2 min followed by liquid nitrogen for 1 minute. Following freeze thaw cycles, samples were completely thawed and incubated at room temperature for 5 minutes. 50 ul of chloroform was added to each sample, vigorously vortexed for 30 s and incubated at room temperature for 3 minutes. Then, samples were centrifuged at 4°C for 15 minutes at 12,000xg for 15 min. Following the spin, 125 ul of the upper aqueous phase was transferred to a fresh 1.5 ml tube. 187.5 ul of 100% molecular grade ethanol was then added to each sample and pipetted up and down to mix. Samples were then transferred to Qiagen RNeasy Mini Kit columns, centrifuged at 8,000xg for 30 seconds, and flow through discarded. Then, 700 ul of RW1 buffer was added to column, centrifuged at 8,000xg for 30 seconds, and flow through discarded. 500 ul RPE buffer added to column, centrifuged at 8,000xg for 30 seconds, and flow through discarded. Then again 500 ul of RPE buffer was added, centrifuged at 12,000xg for 2 min, and flow through discarded. Columns were then transferred to fresh collection tubes, centrifuged at 13,000xg for 1 minute with tube lids open to dry columns. Samples were then eluted from column with 20 ul of RNase free water into a fresh 1.5 ml microcentrifuge tube by centrifuging at 13,000xg for 1 min. Following RNA extraction, RNA samples were DNase treated with Turbo DNA-free Kit (Invitrogen #AM1907) following manufacturer's directions. Briefly, 2 ul of 10x Turbo DNase Buffer and 1 ul of Turbo DNase were added to 20 ul RNA samples and gently mixed by pipetting up and down. Samples were then incubated in a 37°C bead bath for 20 min. Following incubation, 3 ul of resuspended Inactivation Reagent was added to each sample and mixed well by pipetting up and down. Samples were incubated at 25°C for 5 min. Then, samples were centrifuged at 10,000xg for 90 sec. The RNA containing supernatants were transferred to fresh tubes and stored at -80°C until RNA sequencing. Stranded mRNA library preparation was conducted using NEBNext® Poly(A) selection following manufacturer's instructions.