GSM3608698: AM21_Q35_EV_2; Caenorhabditis elegans; RNA-Seq
Total RNA was isolated using NucleoSpin® RNA kit (MACHEREY-NAGEL, 740955). On each time-point, we washed the worms with M9 to remove bacteria, and resuspended them in 350 µl buffer RA1 (kit reagent), 5.5 µl 1M DTT, and a small amount of 0.5 mm zirconium oxide beads (Next Advance, ZrOB05). The worms were homogenized using a Bullet Blender® (Next Advance) set to speed eight, for three minutes, and then set to speed nine and for an additional two minutes. Homogenates were transferred to fresh tubes and centrifuged at 14,000 x g for 5 minutes. The supernatant was transferred to NucleoSpin® Filter (kit reagent), and the next steps followed the manufacturer's standard protocol. Following RNA isolation, the RNA integrity and quantity was assessed using the Agilent 2200 TapeStation System. The RNA purity was assessed by loading 1.5 µl of each sample onto a NanoDrop™ 2000c Spectrophotometer and reading the A260 nm emission, as well as the A260/A280 and A260/A230 ratios. Isolated RNA samples were prepared and sequenced by the Technion Genome Center (Technion, Haifa, Israel) according to the CEL-Seq2 protocol, as described by its developers (Hashimshony et al., 2016).
GEO Accession
GSM3608698