title: GSM1643204: PGCd51_5; Macaca fascicularis; RNA-Seq
library_construction_protocol: Total RNA was extracted from hiPSCs, iMeLCs, and hPGCLCs or mESCs, mEpiLCs, and mPGCLCs by using an RNeasy Micro Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.  Synthesis and amplification of cDNAs using 1 ng of purified total RNA, and construction of cDNA libraries for RNA sequencing (ABI SOLiD 5500XL system; Life Technologies) were performed as described in (Nakamura et al., 2015). For isolating single cells of cyESCs for the SC3-seq analysis [Nakamura et.al., Nuceic Acids Res. (2015)], cells were first detached as clumps by the CTK solution [0.25% of Trypsin (Life Technologies), 0.1 mg/mL of Collagenase Ⅳ (Life Technologies), 1 mM of CaCl2 (Nacalai Tesque)], were incubated in 0.25% of trypsin/PBS (Sigma-Aldrich) for around 10 min at 37℃, and were then dispersed into single cells in 1% (vol/vol) KSR/PBS. Genital ridges were dissected out and were dissociated into single cells by incubating with 0.25% trypsin/PBS for around 10 min at 37℃ followed by repeated pipetting. The resulting single cells were dispersed in 0.1 mg/ml of PVA/PBS (Sigma-Aldrich) and were processed for the SC3-seq analysis (Nakamura et al., 2015). cDNA synthesis and amplification from isolated RNAs/single cells were perfomed essentially as reported in [Kurimoto, K.et.al., Nucleic Acids Research, 34(5), e42.]. Library constructions for SOLiD System were performed as reported in [Nakamura et.al., Nuceic Acids Res. (2015)].
attributes.tag: GEO Accession
attributes.value: GSM1643204