GSM4500947: Rep2_ExpressingProgeny_SmallRNAs; Caenorhabditis elegans; ncRNA-Seq
RNA extraction was performed as previously described (Posner et al., 2019): For RNA isolation worms were lysed using the TRIzol reagent. 300 μL of TRIzol was added to 50ul of adult worms and were subjected to three cycles of freezing in −80C and vortexing at RT for 15mins. 60 μL of chloroform was added and samples were transferred to a pre-spun Heavy Phase Lock tube and centrifuged at 12,000 g for 15mins at 4C. The aqueous phase was transferred to a pre-spun Heavy Phase Lock tube and 1:1 Phenol:Chloroform:Isoamyl Alcohol was added and centrifuged at 16,000 g for 5mins at RT. The aqueous layer was transferred to a 1.5ml Eppendorf tube and 20 μg of Glycogen and 1:1 Isopropanol was added. The samples were incubated at −20C for 30 mins and then centrifuged for 30mins at 16,000 g at 4C. The pellet was washed 2 times with 70% ethanol and then air-dried for 10 minutes. The pellet was re-suspended in 10ul of RNase free water.  Small RNA libraries: total RNA samples were treated with tobacco acid pyrophosphatase (TAP, Epicenter), to ensure 5′ monophosphate-independent capturing of small RNAs. Libraries were prepared using the NEBNext® Small RNA Library Prep Set for Illumina® according to the manufacturer's protocol. The resulting cDNAs were separated on a 4% agarose E-Gel (Invitrogen, Life Technologies), and the 140–160 nt length species were selected. cDNA was purified using the MinElute Gel Extraction kit (QIAGEN). Libraries were sequenced using an Illumina HiSeq 2500 instrument.