GSM8711659: RNA-TAO-ctr-1; Homo sapiens; RNA-Seq
RNA-seq:Cells were dissociated with Trypsin for 5 min, stopped with serum containing media, total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer's instructions.Embryos (five embryos per sample) were collected in a 0.2-mL PCR tube, and 4 µL lysis buffer (0.2% Triton X-100, RNase inhibitor, dNTP, and oligo-dT primers) was added. The samples were immediately incubated at 72 °C for 3 min, and cDNA was produced using SuperScript II reverse transcriptase. The RNA-seq transcriptome librariy of cell was prepared following Illumina® Stranded mRNA Prep, Ligation from Illumina (San Diego, CA) using 1μg of total RNA. Embryos RNA-seq libraries were generated from early mouse embryos using the Smart-seq2 protocol.The cut-tag library of cells were made as following steps: treated with 10 μl of pre-washed ConA beads in a 1.5-ml low-binding tube. Next, 50 μl of antibody buffer containing 0.5 μg of antibody was added, and the mixture was incubated for 2 hours at room temperature. After two washes with dig-wash buffer, 50 μl of dig-wash buffer with 0.5 μg of secondary antibody was added, followed by a 30-minute incubation at room temperature. The samples were washed twice with 800 μl of dig-wash buffer, then 0.58 μl of pG–Tn5 and 100 μl of dig-300 buffer were added. After a 1-hour incubation at room temperature, another two washes with 800 μl of dig-wash buffer were performed. Tagmentation was carried out by adding 300 μl of tagmentation buffer, and the samples were incubated at 37 °C for 1 hour. The reaction was halted by adding 10 μl of 0.5 M EDTA, 3 μl of 10% SDS, and 2.5 μl of 20 mg/ml Proteinase K. Following phenol-chloroform extraction and ethanol precipitation, PCR was conducted to amplify the libraries, which were sequenced using Illumina Hi-Seq Xten or Hi-Seq 2500 systems, following the manufacturer's guidelines.