GSM7035482
PBMCs from the recipients of CAR19/IL-15 NK cell therapy were enriched by Ficoll-hypaquse density gradient centrifugation, kept frozen in the vapor phase of liquid nitrogen, and thawed from the time points as indicated. Thawed cells were rested for 20 min in a humidified incubator at 37 degree Celsius in the presence of 5% CO2, washed in PBS with 1% BSA, and stained for a viability dye (Tonbo BioScience), CD19 (Clone HIB19: BD Bioscience), CD33 (Clone P67.6: BD Bioscience), CD14 (Clone M5E2: BD Bioscience), CD3 (Clone SK7: BD Bioscience),CD20 (Clone 2H7: BD Bioscience), CD56 (Clone N901: BeckMan Coulter), CD45 (Clone J33: BeckMan Coulter). CAR was detected on donor HLA positive, CD3- , CD16+ and CD56+ cells by using the fluorophore-tagged F(ab)2 fragment against Human IgG (Jackson Immunoresearch) as described previously . Single cells from the donor NK cell population, or from B cells of the patients were sorted into each well of a 96 well plate with lysis buffer (10 X l of TCL buffer, Qiagen and 1% 2-mercaptoethanol: Sigma). The plates were spun down at 400xg for 5 minutes at 4 degree Celsius and immediately stored at -80°C until library preparation. cDNA was synthesized as described by the Smart-Seq2 protocol from Picelli et al.. Briefly, RNA was purified using RNAClean XP beads (Beckman Coulter) and reverse transcribed to cDNA using Maxima RNAse H-minus (ThermoFisher) in the presence of biotinylated oligo-dT30VN, biotinylated template-switching oligonucleotides, and betaine. Next, a PCR pre-amplification step was done with KAPA HiFi HotStart Ready Mix (Roche Diagnostics) and biotinylated ISPCR primers. DNA products were further purified with Agencourt XP DNA beads, and the concentration of each sample was quantified by Qubit dsDNA HS Assay kits (Invitrogen). Quality of samples was analyzed on a 2100 Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). 0.15 ng of each sample was tagmented and Nextera index adapters were used for barcoding according to the Illumina Nextera XT DNA sample preparation kit (Illumina).