GSM5746613: Hypodermal SLAM-ITseq rep2; Caenorhabditis elegans; RNA-Seq
Samples were lysed in 5 freeze/thaw cycles (1 min in liquid nitrogen; 3 min in 37°C shaker) and left for 3 min at RT. Then, 100 µl chloroform was added, tubes were shaken vigorously for 15 seconds and left for 3 min at RT. After centrifugation at 12000 g, 4ºC for 15 min, the aqueous phases (~300 µl) were transferred to new tubes containing 0.5 µl 0.1 M DTT and 0.5 µl 20 mg/ml glycogen. RNA was precipitated by adding 250 µl isopropanol and centrifugation at 12000 g, 4ºC for 10 min. Supernatants were removed and pellets were washed in 500 µl EtOH 75%. After centrifugation and removal of the supernatants, the pellets were left to dry for 5 min and resuspended in 20 µl 1 mM DTT. After incubation at 55ºC for 10 min, residual DNA was digested by treatment with DNase I (New England Biolabs) in a total volume of 50 µl at 37ºC for 10 min. DNase I was inactivated by addition of 0.5 µl 0.5 M EDTA. RNA was purified with Monach spin columns (New England Biolabs), eluted in 20 µl 1 mM DTT and evaluated on a Nanodrop. Alkylation of RNA was performed by incubating 1.5 µg RNA with 25 µl DMSO, 5 µl 0.1 M iodoacetamide (Sigma-Aldrich) and 5 µl 0.5 M sodium phosphate (pH 8.0) in a total volume of 50 µl at 50ºC for 15 min. The reaction was stopped by adding 1 µl 1M DTT. RNA was precipitated by adding 0.5 µl 20 mg/ml glycogen, 5 µl 3 M sodium acetate (pH 5.2) and 180 µl 96% ethanol followed by incubation at -20ºC for 20 min and centrifugation at 15000 g, 4ºC for 20 min. The supernatants were removed and the pellets were washed in 180 µl 75% ethanol followed by centrifugation at 15000 g, 4ºC for 5 min. Again, supernatants were removed and pellets were left to dry for 5 min. RNA was resuspended in 15 µl H2O, incubated at 55ºC for 8 min and evaluated on a Nanodrop. NGS libraries were prepared at EMBL GeneCore using a QuantSeq 3′ mRNA-Seq Library Prep Kit for Illumina (Lexogen).