title: GSM1909760: E10.5_PGC_2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNAs from collected primordial germ cell were extracted with RNeasy Micro Kit (QIAGEN, 74004) according to the manufacture's instruction. 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.] The numbers of PCR cycles were employed for 11 cycles. 5 ng of amplified and quality-checked cDNAs were added to the pre-amplification buffer [1 × ExTaq buffer [Takara Bio (RR006), Shiga, Japan], 0.2 μM of each dNTP [Takara Bio (RR006)], 0.01 μg/μl of the N-V3 (dT)24 primer (HPLC-purified, attachment of amine at the 5-prime end), 0.01 μg/μl of the V1(dT)24 primer (HPLC-purified), and 0.025 U/μl of ExTaqHS [Takara Bio (RR006)]], and were amplified by four cycles of PCR. The surplus primers and PCR byproducts such as primer dimers were removed by size selection through three rounds of purification using a 0.6 × volume of AMPureXP beads for each  round [Beckman Coulter (A63881), Tokyo, Japan] according to the manufacturer’s instructions. The purified cDNAs were diluted to 130 μl by double-distilled water (DDW) and fragmented by shearing with Covaris S2 or E210 (duty cycle: 10%; intensity: 5; cycles per burst: 200; duration: 50 seconds; an intensifier was installed in the case of E210) [Covaris, Woburn, MA], and then end-polished in the End-polish buffer [1 × NEBnext End Repair Reaction buffer [NEB (B6052S), Ipswich, MA], 0.01 U/μl of T4 DNA polymerase [NEB (M0203)], and 0.033 U/μl of T4 polynucleotide kinase [NEB (M0201)]] for 30 min at 20oC. After incubation, a 0.8 × volume of the AMPureXP was immediately added, the solution was mixed for more than 20 min, and then the supernatant was transferred to a 1.2 × volume of the AMPureXP reagent and the cDNAs were purified. Next, to provide the purified cDNAs with an Int-adaptor sequence, the cDNAs were incubated in 30 μl of the Internal adaptor extension buffer [1 × ExTaq Buffer, 0.23 mM of each dNTP, 0.67 μM of the IntV1 (dT)24 primer (HPLC-purified), 0.033 U/μl of ExTaqHS] using the following thermal cycler program: 95℃ for 3 min, 67℃ for 2 min, and 72℃ for 2 min. The reactions were terminated by chilling in an ice-block, and after the addition of 20 μl of the P1-adaptor ligation buffer [a mixture of 10 μl of 5 × NEBNext Quick Ligation Reaction Buffer [NEB (B6058S)], 0.6 μl of 5 μM of the P1-T adaptor [Life Technologies (4464411)], and 1 μl of T4 ligase [NEB (M0202M)]], the solution was incubated for 15 min at 20℃ and for 20 min at 72℃. After two rounds of cDNA purification by adding a 1.2 × volume of AMPure XP, the cDNAs were added into the Final amplification buffer [1 × ExTaq buffer, 0.2 mM of each dNTP, 1 μM of the P1 primer, 1 μM of the BarT0XX_IntV1 primer (HPLC-purified), 0.025 U/μl of ExTaqHS] and amplified by PCR using the following thermal cycler program: 95oC for 3 min; followed by 9 cycles of 95℃ for 30 sec, 67℃ for 1 min and 72℃ for 1 min; with a final extension of 72℃ for 3 min. Finally, the cDNA libraries were purified two times by using a 1.2 × volume of AMPureXP and dissolved in 20 μl of TE buffer. The quality and quantity of the constructed libraries were evaluated by LabChip GX or Bioanalyzer 2100, a Qubit dsDNA HS assay kit [Life Technologies (Q32851)], and a SOLiD Library TaqMan Quantitation kit [Life Technologies (4449639)]. The cDNA libraries were sequenced with an ABI SOLiD 5500XL system (Applied Biosystems) for 50-bp single-end reads according to the instructions of the manufacturer.