GSM7143066: 1110A; Mus musculus; RNA-Seq
To isolate CD4+ T cells, mouse dorsal skin was excised and the fat layer was removed using a surgical scalpel. The skin tissue was treated with dispase II (2.5 mg/ml) in Hank's Balanced Salt Solution for 60 min at 37 °C. The dermal fraction was collected by scraping and then cut into small pieces. Dermal single-cell suspensions were obtained after collagenase IV (1 mg/ml) treatment for 2 h at 37 °C, followed by filtering and centrifugation. Samples were stained for 30 min on ice. The following antibodies were used: anti-CD4-PE (100408, Biolegend) and anti-CD45-FITC (103108, Biolegend). According to the sample and product requirements, select the corresponding detection method for quality inspection, take a sample no larger than 4 μL, add Oligo-dT Primer, place it on a PCR instrument, and perform RNA denaturation; prepare a reverse transcription system, and use SMART amplification technology to synthesize the first Strand cDNA. Prepare a PCR amplification system for pre-amplification of cDNA; select the corresponding detection method for quality inspection of cDNA; use the transposase method for qualified amplification products for subsequent library construction; select the corresponding detection method according to product requirements for quality inspection of the library After the PCR product is denatured into a single strand, prepare a circularization reaction system and set up a reaction program to obtain a single-stranded circular product and digest the uncircularized linear DNA molecule.