title: GSM1698491: E95PGCs_2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from E9.5 endogenous PGCs, iPGCs, PGCLCs and EGCs using Qiagen RNeasy Micro Kit (Qiagen) and eluted in 8 μl of RNase free water. 2 biological replicate samples were used with following start cell numbers: E9.5 PGCs (about 250 cells), iPGCs (1500 cells), PGCLCs (300-500 cells) and EGCs (500 cells). RNA was amplified and converted to cDNA using the Ovation RNA-Seq System V2 (Nugen). cDNA was diluted to 130 μl using TE buffer (Tris pH 8.0 10mM, Cat#15568-025; EDTA 0.1mM, Cat#15575, both from Invitrogen) transferred to Snap-Cap MicroTUBEs with AFA fiber (Covaris, Cat# 520015) and sonicated to DNA fragments within the 200bp range using a Covaris S2 sonicator according to manufacturer’s instructions (Duty cycle 10%, Intensity 5, Cycles per burst 200 and time 180 sec). Subsequently libraries were generated using the Encore Rapid Library System according to manufacturers instructions. Library concentration was estimated using a Kapa library quantification kit (Cat# 4827) according to manufacturer’s instructions on a ViiA 7 Real-time PCR System (Invitrogen). Libraries were run using 50bp single-end reads on the HiSeq 2500 System (Illumina). 8 libraries were multiplexed and run in two lanes. ERCC RNA Spike-In Mix (Cat#4456740, Invitrogen) was used according to manufacturer’s instructions.
identifiers.namespace: GEO
identifiers.id: GSM1698491