GSM5517312: MFA_sh346-70; Macaca fascicularis; RNA-Seq
Isolation of single cells. The embryos were washed in PBS for 3 times, washed in 0.25% trypsin (T4799; SigmaAldrich) 2 times, incubated with 0.25% trypsin for around 15 min at 37 °C, and terminated by DFBS. Embryo was dissociated into single cells by repeated pipetting and dispersed in 1% DFBS/PBS. A single cell was pipetted into a PCR tube. All operations above were performed under the Nikon SMZ645 microscopy. The synthesis and amplification of full-length cDNAs were performed following Smart-seq2 protocol4. Briefly, single cells were washed in DPBS (Gibco 14190-144) and picked into lysis buffer using Pasteur pipettes under a dissecting microscope. Reversetranscription reactions and pre-amplification were performed using SuperScript II (Invitrogen 18064-014) and KAPA HiFi HotStart ReadyMix (KAPA Biosystems KK2601), respectively. The quality of the cDNAs were evaluated by Bioanalyzer 2100. Library construction and sequencing were performed by Annoroad Gene Technology (http://www.annoroad.com/). Pair-end sequencing was performed on Illumina X-ten platform.