GSM5952010: wt_l1pgcs_rep01; Caenorhabditis elegans; RNA-Seq
Single sets of 2 Primordial Germ Cells (PGCs) or 2 Early Germ Cells (EGCs) were dissected from L1 larvae or L2 larvae, respectively. PGCs and EGCs were used directly as starting material for library preparation. 20-60 gonads were dissected from 1st day adult hermaphrodites and then cut at the gonad bend. Only the distal halves were kept to exclude oocytes and several somatic gonad cells from our analysis. Gonads were vortexed in Trizol and stored at -80˚C. RNAs were then extract from those gonads using 1-bromo-3-chloropropane (Sigma #B9673), precipitated using isopropanol, pelleted by a 13,000 rcf spin for 15 minutes at 4˚C, and washed using ethanol. Libraries were constructed according to manufacturer's recommendation. PGCs/EGCs: RNAs were converted into double-stranded cDNAs using a modified smart-seq2 protocol (Picelli et al., 2014). Illumina sequencing libraries were then prepared from those cDNAs using Illumina's Nextera XT kit. Gonads: mRNAs were isolated from total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and then used to prepare Illumina sequencing libraries using the NEBNext Ultra RNA Library Prep Kit (#E7530).