title: GSM6661528: ag35AG_1_MS257_020; Macaca fascicularis; RNA-Seq
library_construction_protocol: Total RNA extraction from 5000-20000 cells were carried out using Nucleospin RNA XS extraction kit (Takara, 740902). cDNA synthesis was performed as described previously (Nakamura et al, 2015). Briefly, 0.2ng/µL of V1(dT)24 sequence was annealed to the 3' end of the 1ng of total RNA by incubation at 70ºC for 90 seconds. Reverse transcription was performed by adding 133U/µL of SuperScript III (Invitrogen, 1089075) and serial incubation at 50ºC for 5 min and at 70ºC for 10 min. The residual primers were excised by treating with 0.5U/µL of Exonuclease I (Takara, 2650A) at 37ºC for 30 min followed by 80ºC for 25 min. Poly-A was added to the 3' end of the cDNA by incubating at 37ºC 15 min and 70ºC for 10 min in the presence of 3mM dATPs (GE Healthcare, 28-4065-01) and 0.75U/µL terminal nucleotidyl transferase (TdT, Invitrogen, 10533-065). For cDNA amplification, the cDNA was applied to thermal cycling in the presence of 0.62 µM of V3(dT)24 and V1(dT)24, 0.05U/µL Ex Taq HS (Takara, RR006).