GSM3209296: ESC_Tbx1RFPB4; Mus musculus; RNA-Seq
Mouse embryos (E7.75¬) were dissected using forceps under a stereomicroscope (Zeiss) and regions of interest were dissociated and harvested using TrypLE. Embryoid bodies (EBs) and cells were dissociated and harvested using TrypLE. Single-cells were analyzed for RFP/GFP expression or sorted using a SH800 Cell sorter (Sony Biotechnologies). GFP+ and RFP+ cells were isolated using a SH800 cell sorter (Sony Biotechnologies) into 96 plates containing water (2.4 mL) with RNase-free DNase I (0.2 mL; NEB) and RNase inhibitor (0.25 mL; NEB). Each sample represents 10 cells. DNase I was inactivated by increasing the temperature (72 C for 3 min), and samples were then stored on ice. Custom-designed 2A oligo 1-mL primer (12 mM, Integrated DNA Technologies was added and annealed to the polyadenylated RNA by undergoing a temperature increase (72 C for 2 min) and being quenched on ice. A mixture of 1 mL of SMARTscribe reverse transcriptase (Clontech Laboratories), 1 mL of custom-designed TS oligo (12 mM, Integrated DNA Technologies , 0.3 mL of MgCl2 (200 mM, Sigma), 0.5 mL of RNase inhibitor (Neb), 1 mL of dNTP (10 mM each, Thermo), and 0.25 mL DTT (100 mM, Invitrogen) were incubated at 42 C for 90 min, which was followed by enzyme inactivation at 70 C for 10 min. A mixture of 29 mL of water, 5 mL of Advantage2 taq polymerase buffer, 2 mL of dNTP (10 mM each, Thermo), 2 mL of custom-designed PCR primer (12 mM, Integrated DNA Technologies, and 2 mL of Advantage2 taq polymerase was directly added to the reverse transcription product, and the amplification was performed for 19 cycles. The amplification product was purified using Ampure XP beads (Beckman-Coulter). Libraries and transposome assembly were made using a previously published protocol. Briefly, 100 pg of total cDNA was added to a 2x tagment DNA Buffer (TD) (2x TAPS buffer: 20 mM TAPS-NaOH, 10 mM MgCl2 (pH 8.5) at 25 C, and 16% weight volume (w/v) PEG 8000), and then spiked with 0.5 mL of 1:64 diluted Tn5 (Epicenter) and incubated for 8 min at 55 C. Tn5 was stripped off from the tagmented DNA by adding 0.2% SDS for a final concentration of 0.05%. Libraries were enriched used KAPAHiFi, which included 5X Kappa Fidelity Buffer, 10 mM dNTPs, and HIFI polymerase, and 1 uL of index primers was used directly in the enrichment PCR amplification of libraries for the Illumina sequencers for a 50-mL reaction. The PCR program was as follows: 5 min at 72 C and 1 min at 95 C, and then 16 cycles at 30s at 95 C, 30s at 55 C, 30s at 72, and 5 min at 72.