GSM4500959: Rep4_ExpressingProgeny_mRNA; Caenorhabditis elegans; RNA-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.  mRNA libraries: cDNA was generated using SMART-Seq v4 Ultra Low Input RNA (Takara) and libraries were prepared using Nextera XT DNA Sample Preparation Kit (Illumina). Libraries were sequenced using an Illumina MiniSeq instrument.