title: GSM2187978: E10.5 Tet1-KO PGC RNA-Seq Replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: For study of Tet1-KO PGCs, total RNA from sorted PGCs isolated from individual Tet1-KO or wild type embryos was isolated using ZR-Duet DNA-RNA MiniPrep kit (Zymo), and RNA from between two to six embryos (equivalent to 1,000 to 8,000 cells) of the same genotype, stage and sex was pooled and concentrated to 6 µL final volume using the RNA Clean and Concentrator 5 kit (Zymo). For study of wild type PGCs isolated from embryos produced by crossing MF1 females with GOF18ΔPE-EGFP males, total RNA from 600-1,000 sorted E10.5 PGCs was isolated using the Nucleospin RNA XS kit (Macherey-Nagel). cDNA synthesis and amplification (15 cycles) was performed with the SMARTer Ultra Low Input RNA kit (Clontech) using between 100 pg and 3ng total RNA and following the manufacturer’s instructions. The amplified cDNA was fragmented by a Covaris S2 sonicator (Covaris) and following the manufacturer’s instructions. Sheared cDNA was converted to sequencing libraries using the NEBNext DNA Library Prep kit (NEB), following the manufacturer’s instructions and using 15 cycles of amplification. The libraries were purified by AMPure XP beads (Beckman-Coulter) and sequenced on the Illumina HiSeq 2500 instrument.