GSM4704499: 0503L12b: Chimpanzee iPSCs rep 2; Pan troglodytes; RNA-Seq
The rhesus macaque blastocysts on day 5 post IVF was used for immunosurgery to remove zonae pellucidae and trophectoderm as explained elsewhere(Mitalipov et al., 2006). In summary, the zonae pellucidae of rhesus blastocysts will be removed by exposure to 0.5% pronase in TH3 medium for 45 seconds. For immunosurgical isolation of rhesus ICM to remove trophectoderm, zona pellucidae-free rhesus blastocysts were exposed to rabbit anti-rhesus spleen serum for 30 minutes at 37°C from VandeVoort laboratory at UC Davis. Subsequently, the blastocysts were washed in TH3 medium for 3 times and were incubated in guinea pig complement reconstituted with HECM-9 (1:2 vol/vol) at 37°C for 30 minutes(Mitalipov et al., 2006). After the incubation, the trophectoderm was removed mechanically using a small-bore pipette (125μM inner diameter) and the clumps of ICM cells from each blastocyst were washed in PBS and placed in a separate PCR tube containing 5μL of RNAse-free water with 5% RNAse inhibitor. The ICMs were quickly frozen on dry ice and were stored at -80°C for cDNA synthesis and RNA-seq library preparation. The cDNA synthesis and RNA-seq were conducted directly from ICM cells using SMART-Seq V4 Ultra Low input cDNA synthesis kit (Clonetech Inc.). The cDNA concentration was measured using Quibit kit and Bioanalyzer High-Sensitivity DNA kit (Agilent Inc.). The cDNA from each ICM were used for library preparation following Illumina Nextera XT kit protocol (Illumina Inc.). The quality and the quantity of the final library were evaluated using Bioanalyzer High-Sensitivity DNA kit (Agilent Inc.),Kappa kit, and the Agilent 2100 Bioanalyzer instrument. Dual index RNA-seq library from ICMs were pooled together with dual index RNAseq libraries of approximately 50 iPSCs for sequencing with dual index Illumia platform on a HiSeq 4000 sequencing machine. The low input cDNA synthesis and RNA-seq library preparation for ICMs and iPSCs were performed simultaneously on similar number of cells and pooled together to be sequenced in the same batch to minimize the potential batch effects. The sequencing coverage approximately 50 million reads per RNA-seq sample.