Ampure xp magnetic beads

Purified mtDNA from MWCNT-exposed BECs were sequenced for the appearance of heteroplasmies and indel mutations compared to the sequences of vehicle-treated control cultures. Tagmentation of mtDNA (combined short and long fragments) was carried out using a Nextera DNA Library Prep kit (Illumina) according to the manufacturer’s instructions. DNA samples were added to a 96-well thermal cycler plate with Amplicon Tagment Mix and Tagment DNA buffer from the kit and heated at 55°C for 5 minutes. Libraries were then amplified with indexed adapters and Nextera PCR Master Mix, first denaturing at 72°C and then running 15 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. AMPure XP magnetic beads (Illumina) were used to purify the resulting mtDNA libraries, according to manufacturer instructions. AMPure beads were added to each sample and allowed to incubate at room temperature for 7 minutes on a magnetic plate to pellet the DNA/beads. Supernatants were discarded and the bead pellets were washed twice with 80% ethanol, then allowed to air dry completely (approximately 10 minutes). Purified DNA was resuspended in AMPure Resuspension Buffer and the DNA concentration/quality was verified by QuBit High-Sensitivity Analysis (Thermo-Fisher) and the Agilent Bioanalyzer.

TCR sequencing was performed using the Mouse TCR beta, Illumina, V-C genes kit from iRepertoire, Huntsville, AL. In brief, 500 ng of RNA was reverse transcribed using a One-Step reverse transcription and amplification kit (Qiagen). The PCR product was purified using Ampure XP magnetic beads (Agencourt), and amplified again (TopTaq PCR Kit, Qiagen), to add adaptor sequences. Libraries were purified with Ampure XP magnetic beads and sequenced using Illumina MiSeq 150 nt paired-end read-length. The TCR CDR3 sequences were extracted from the raw sequencing data by iRepertoire. Briefly, raw paired-end fastq files were first demultiplexed based on barcode and merged reads were mapped using a Smith-Waterman algorithm to germline V, D, J and C reference sequences from the IMGT web site (http://www.imgt.org). To define the CDR3 region, the position of CDR3 boundaries of reference sequences from the IMGT database were migrated onto reads through mapping results and the resulting CDR3 regions were extracted and translated into amino acids. Reading frames not containing a stop codon were filtered and error-corrected using iRepertoire proprietary SMART algorithm.

CD25⁺FoxP3(GFP)⁺CD4⁺ Tregs were sorted into RL buffer (Norgen, 51800) from the omenta and spleens of 4 naïve and 4 day 15 EG7.1.15 tumor-bearing FoxP3-GFP-DTR mice. Sorted cells were not pooled, thus each sample represents a Treg population from an organ of an individual mouse (Supplemental Table 2). Total RNA was purified using Single Cell RNA purification kit (Norgen) according to the manufacturer’s instructions. TCR library was prepared using iRepertoire arm-PCR platform (iRepertoire) as previously described (31 (link)). Briefly, reverse transcription of RNA was conducted with OneStep RT-PCR kit (Qiagen, 210212) according to the manufacturer’s protocol. The PCR product was purified using Ampure XP magnetic beads (Agencourt, A63880), and secondary amplification of the resulting product was performed (GoTaq PCR Kit, Promega, M7660), allowing addition of Illumina adapter sequences. Finally, libraries were purified with Ampure XP magnetic beads and sequenced using Illumina MiSeq 250 nt paired-end read-length. The TCR CDR3 sequences were extracted from the raw sequencing data, wherein fastq files were first demultiplexed and reads were then mapped to germline V, D, J and C reference sequences from the IMGT using iRepertoire bioinformatics tools.