GSM2690485: Wild-type embryonic PGC transcriptome WT_emb_25_Slee78; Caenorhabditis elegans; RNA-Seq
Worm were dissociated with pronase solution. Cell suspensions were subjected to FACS to isolated PGL-1::GFP marked primordial germ cells (PGCs). Total RNAs were extracted using Trizol reagent and RNA purifiaction was done using RNA clean and concentrator-5 (Zymo researh). Three different RNA-seq library preparation methods were used for this study.SMART-seq libraries: libraries were made from 2ng of total RNA isolated from sorted PGCs from worms grown at 25oC using SMART-seq v4 Ultra Low input RNA kit (Clontech, Cat. No. 634888 ) followed by Low Input Library Prep Kit (Clontech, Cat. No. 634947). The cDNAs were then fragmented using Covaris AFA system at the Johns Hopkins University microarray core and cloned using the Low Input library prep Kit. Nugen Ovation libraries: libraries were made from 3ng of total RNA isolated from sorted cells from worms grown at 25oC using Nugen Ovation system V2 (# 7102-08) followed by Nugen Ultralow library system. TruSeq libraries: 50ng of total RNA isolated from sorted PGCs from L1 worms grown at 20oC were subjected to Ribozero kit (illumina, MRZE706) to remove rRNA. Libraries were constructed using Truseq Library Prep Kit V2.