Solexa hiseq 2000 platform

The IgAN susceptibility loci discovered in genome-wide association studies (GWAS) were primarily from five genomic regions: the MHC region at 6p21, the DEFA locus at 8p23, the TNFSF13 locus at 17p23, the HORMAD2 locus at 22q12 and the CFH/CFHR locus at 1q32 (10 (link), 11 (link)). Thus all of the SNPs reported as top association signals in GWASs of IgAN were selected for further testing in LN. In total, 15 SNPs, including 8 SNPs within the MHC loci and seven SNPs outside the MHC region, were genotyped. In the discovery cohort from Beijing, genotyping in LN patients was performed with an Illumina Solexa HiSeq 2000 platform, and selected SNPs checked in the SLE patients without LN were genotyped using TaqMan allele discrimination assays (Applied Biosystems, Foster City, California, USA). For replicates, genotypes were determined using TaqMan allele discrimination assays (Beijing and Shanghai) or were directly retrieved from the original GWAS data (Hong Kong) (9 (link)).

After the total RNA extraction and DNase I treatment, magnetic beads with Oligo-dT were used to isolate Poly (A) mRNA. The mRNA was fragmented into short fragments by the fragmentation buffer. First-strand cDNA was synthesized using fragmented mRNA templates and random hexamer primers. Subsequently, we synthesized second-strand cDNAs using DNA polymerase I, RNaseH, dNTPs, and buffer. We purified short fragments, resolved them with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments were connected with adapters. The suitable fragments were selected as templates for PCR amplification via agarose gel electrophoresis. We used 2100 Bioanaylzer (Agilent, USA) and StepOnePlus Real-Time PCR System (ABI, USA) to quantify and qualify the sample libraries. Finally, the two libraries could be carried out high-throughput transcriptome sequencing using Illumina-Solexa HiSeq^™ 2000 platform.