Iscan system

Extraction of genomic DNA was performed by the phenol-chloroform method [20 ], after which it was bisulfite converted using the EZ DNA Methylation – GoldTM kit (ZymoResearch, USA). The bisulfite converted DNA was hybridized to the Illumina 450 k methylation chip (Illumina, San Diego, CA, USA) to measure DNA methylation in 485 577 individual CpG sites. In the discovery group, DNA obtained from blood samples taken from the same individual before and six months after surgery were hybridized to the same physical chip. The array was imaged using the Illumina iScan system (Illumina, San Diego, CA, USA), determining the percentile methylation for each CpG site across the study groups.

Genomic DNAs were extracted from the peripheral blood lymphocytes of the patients and unaffected controls, using the Wizard Genomic DNA Purification Kit (Promega, WI, USA), according to the manufacturer’s protocol. A whole-genome genotype scan was performed using about 200 ng of the genomic DNAs on Illumina’s HumanOmni1-Quad BeadChip (Illumina, San Diego, USA), according to the manufacturer’s protocol. All samples were scanned using the Illumina iScan system (Illumina), and the normalized bead intensity data were loaded into the GenomeStudio software (Illumina). Considering a potential association between rare variants and rare diseases such as HSCR, this study included all polymorphic markers for association analysis. For 1,140,419 markers on the chip, SNP marker quality control (QC) was applied as follows: (1) only SNP with call rate (>98%, 995,666 markers) in both cases and controls was included, (2) 221,556 monomorphic markers were excluded, and (3) visual inspection of the genotype cluster image was performed for the SNPs with deviation from Hardy-Weinberg equilibrium (HWE, P<0.0001), and 16,850 SNPs deviating from HWE were excluded from the analysis. The 757,260 markers that remained after QC were ultimately used for further association analysis.

Five days after co-culture, CFSE^neg and CFSE^pos LN GC-Tfh cells from HIV-infected and -uninfected individuals were re-sorted into 100ul of cold RLT buffer (Qiagen) supplemented with 1% βM (Sigma Aldrich), and quickly stored at −80°C. Extraction of total RNA followed by DNase I treatment was performed using Qiagen’s RNeasy Micro Kit according to the manufacturer’s protocol. RNA was quantified with a NanoDrop spectrophotometer (Thermo Scientific) and its quality measured using the Experion automated electrophoresis system (Bio-Rad) along with a HeLa RNA positive control and a non-template negative control. RNA was converted into biotinylated cRNA using the Illumina Total Prep-96 RNA amplification kit (Life Technologies). Biotinylated cRNA was normalized and hybridized to the Illumina Human HT-12V4 Expression BeadChips according to the manufacturer’s guidelines, then quantified with an Illumina iScan system (Illumina). Data was collected using Illumina GenomeStudio software.

Five hundred ng of genomic DNA was bisulfite-treated using the EZ DNA methylation kit (Zymo research, Irvine, CA, USA). Although Infinium Mouse Methylation BeadChips have been developed recently to measure the methylation profile of mice, there was no mature mouse methylation array when this study started, and thus, the study was carried out using the Illumina Human Infinium Methylation BeadChips. Bisulfite-converted DNA was hybridized with the Illumina Human Infinium MethylationEPIC BeadChip Kit (Illumina, San Diego, CA, USA) and then scanned with the Illumina iScan System (Illumina, CA, USA) using the manufacturer’s standard protocol. Samples from the two groups were randomly loaded on the arrays.
The Infinium MethylationEPIC BeadChip Kit included >850,000 individual CpG sites genome-wide at single-nucleotide resolution. As the Infinium MethylationEPIC array was not specifically designed for the mouse genome, many primers for each probe in the array do not target or specifically align within the mouse genome, so their location cannot be identified in the mouse genome with certainty.
Hence, 22,569 out of the 850,000 probes that were identified in previous studies [20 (link),21 (link)] to have a unique best alignment score when mapping the probe primers to the mouse genome (GRCm38) were used for the following analysis.