Immunoseq analyser software

Genomic DNA was prepared using DNEasy minicolumns (Qiagen). Sequencing was performed by Adaptive Biotechnologies (Seattle, WA) using the Immunoseq platform and data were analyzed using the ImmunoSEQ Analyser software (Adaptive biotechnologies).
Supplementary Information accompanies this paper.

Intact biopsies from HC and CD patients (with paired inflamed and non-inflamed colon specimens from the latter only) were thawed and enzymatically digested in RPMI supplemented with HEPES, PSG, BCS, MgCl₂, CaCl₂, Collagenase Type 1, and 0.011% DNase for 30 minutes at 37° C. Cells were then pelleted, resuspended, and mechanically digested by passing the suspension through an 8-gauge needle and filtered through a 100-μum screen. Cells were again pelleted, stained as above, and immediately sorted using the BD FACS Fusion. Live single MAIT cells were sorted into a 96-well plate and their TCR alpha and beta chain amplified using a nested-PCR protocol [70 (link)]. PCR products were Sanger sequenced by a commercial third party (GeneWiz; now Azenta Life Sciences, Chelmsford, MA) using primers specific to the common region of the TCR alpha or beta gene, and TCR alignment was performed using the IMGT HighV-quest tool. For blood TCR analyses, up to 50,000 MAIT cells were sorted by FACS as above from thawed PBMC. DNA extracted from sorted MAIT cells was then sent for comprehensive TCR beta chain sequencing by a commercial third party (Adaptive Biotechnologies, Seattle, WA), with data thus generated retrieved from and analyzed on the latter’s proprietary online ImmunoSEQ Analyser software.