title: GSM4552453: Tnaive_2; Mus musculus; ATAC-seq
library_construction_protocol: The following fluorochrom-conjugated antibodies were purchased from BD, Biolegend and eBioscience (human)-CD2 (RPA-2.10), CD4 (RM4-5), Ova‑TCR (KJ1.26), CD62L (MEL14). To prevent unspecific antibody binding, cell suspensions were incubated with 10µg/ml Chrom Pure rat IgG (Jackson ImmunoResearch Laboratories) and 10 µg/ml anti-mouse CD16/CD32 antibody (BioXcell). For surface staining cells were washed and then incubated with the corresponding antibodies dissolved in PBS containing 0.2% BSA for 15 min on ice. Cell sorting for RNA-seq and ATAC-seq analysis was performed at the cell sorting facility (Helmholtz Centre for Infection Research, Braunschweig, Germany) on FACSAria IIu (BD Bioscience) and FACS Aria-II SORP (BD Bioscience). Acquired data was analyzed utilizing FlowJo software (Tree Star). Cells were washed once with PBS before DNA transposition was performed with the Nextera DNA Library Prep Kit (Illumina). Per sample, the following kit components (25 µl TD, 2.5 µl TDE1 and 22µl nuclease free water) were combined and placed at 37°C for 3 min before 0.5µl of 1% Digitonin (Promega) was added to the master mix (64). Samples were resuspended in the transposition reaction mix and incubated for 30 min at 37°C at 300rpm. After transposition DNA purification was carried out with the MinElute PCR Purification kit (Qiagen) according to manufacturer's instructions. Transposed DNA fragments were pre-amplified by combining the following reagents per reaction (5μl transposed DNA, 10μl nuclease free water, 2.5μl 25 μM custom Nextera PCR primer 1, 2.5μl 25 μM custom Nextera PCR primer 2, 25μl NEBNext High-Fidelity 2x PCR Master Mix (New England BioLabs)) and running the following 6-cycle PCR program (1 cycle of 72°C for 5 min, 98°C for 30 sec; 5 cycles of 98°C for 10 sec, 63°C for 30 sec, 72°C for 1 min). The forward primer was identical for all samples 5′-AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG-3′ whereas the reverse primer contained a barcode (underlined) used for demultiplexing 5′-CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT-3′ (65). The appropriate amount of further amplification cycles was determined by qPCR prior to the final sample amplification (45μl of previously PCR amplified DNA, 39.7μl nuclease free water, 2.25μl 25 μM customized Nextera PCR primer 1, 2.25μl 25 μM customized Nextera PCR Primer 2, 0.81μl 100x SYBR Green I, 45μl NEBNext High-Fidelity 2x PCR Master Mix) with the PCR program (1 cycle of 98°C for 30 sec; 8-10 cycles of 98°C for 10 sec, 63°C for 30 sec, 72°C for 1 min). PCR purification was again carried out using the MinElute PCR Purification kit. Finally, size selection was performed with SPRIselect beads (Beckmann-Coulter) according to manufacturers instructions. DNA quality, content and fragment size was assessed with Agilent Technologies 2100 Bioanalyzer profiles and Qubit measurements. Sequencing of libraries was carried out at the sequencing facility on Illumina NOVASeq6000 using 50 bp paired-end reads and quality of sequenced libraries was verified with FastQC.
attributes.tag: GEO Accession
attributes.value: GSM4552453