GSM2722414: pgc2; Xenopus laevis; RNA-Seq
Primordial germ cells and stage-matched endoderm cells were isolated from stage 12-14 Xenopus laevis embryos and collected into RNA lysis buffer as described by Butler et al. (2016). Total RNA was extracted using the RNAqueous-Micro Total RNA Isolation Kit per the manufacturer’s protocol (Ambion). Three 1ng aliquots of each rRNA-depleted sample were used as template for preparation of sequence-able template DNA molecules using the ScriptSeq v2 RNASeq Library Preparation Kit (Illumina, Part # SSV21124). The quality and size distribution of the amplified libraries were determined utilizing an Agilent 2100 Bioanalyzer High Sensitivity DNA Chip. Libraries were quantified using the KAPA Library Quantification Kit (Kapa Biosystems, Boston, MA), and equimolar concentrations were diluted prior to loading onto the flow cell of the Illumina cBot cluster station. The libraries were extended and bridge amplified to create sequence clusters using the Illumina HiSeq PE Cluster Kit v4, and sequenced on an Illumina HiSeq Flow Cell v4 with 100-bp paired-end reads plus index read using the Illumina HiSeq SBS Kit v4. Real time image analysis and base calling were performed on the instrument using the HiSeq Sequencing Control Software version 2.2.58. Three DNA libraries were prepared from Xenopus laevis primordial germ cell RNA and three from matched endoderm cell RNA.