GSM6280408: deca3_day5_coculture_1; Homo sapiens; OTHER
For in vitro experiments, T cells cocultured with NALM-6 cells were harvested at multiple time points after clearance of NALM-6, and genomic DNA was isolated by QIAGEN's DNA purification kit. For in vivo experiments, NSG mice were euthanized, and spleen and bone marrow were collected immediately. Red blood cells were lysed by ACK Lysis Buffer (Thermofisher) for 2 mins. After a wash, cells were analyzed and sorted by BD Facs Aria for CAR+ cells, and then genomic DNA was harvested using QuickExtract DNA extraction solution (Lucigen). The crRNA library readout was performed using a two-step PCR strategy, where the first In-Out PCR was used to amplify out the integrated fragments from gDNA and the second PCR adds appropriate sequencing adapters to the products from the first PCR. For the first round PCR, the thermocycling parameters were 98 °C for 1 min, 20 cycles of (98 °C for 1s, 60 °C for 5s, 72 °C for 25 s), and 72 °C for 2 min. In each PCR reaction, we used 2 µg of total gDNA for in vitro and used 5ul DNA extraction solution for in vivo. A total of 3-4 reactions was used to capture the full representation of the library. PCR products for each biological sample were pooled and used for amplification with barcoded second PCR primers. For the second round PCR, the thermocycling parameters were 98 °C for 1 min, 28 cycles of (98 °C for 1s, 61 °C for 5s, 72 °C for 10s), and 72 °C for 2 min. Second PCR products were pooled and then normalized for each biological sample before combining uniquely barcoded separate biological samples. The pooled product was then gel purified from a 2% E-gel EX (Life Technologies) using the QIAquick Gel Extraction Kit (Qiagen). genome-integrated crRNA readout