Genechip scanner 7g

Total 18 tumor i.e., pooled-OT, OT-3, OT-7, OT-9, OT-33, OT-38, OT-39, OT-40, OT-42, OT-43, OT-44, and OT-45, and 5 oral control i.e., OC-2, OC-6, OC-22, kit-based positive and negative controls, were processed for CNVs. DNA (12 ng/μL per sample) was processed on a MIP-based OncoScan array for CNV profiling. According to the recommended protocol, the chips were processed for hybridization, staining, and washing procedures and were finally scanned through GeneChip Scanner-7G (Affymetrix, Santa Clara, CA, USA) for identification of the copy number and somatic mutation variations as reported previously [44 (link), 51 (link)]. The OSCHP files were generated using OncoScan Console Software (Biodiscovery, Inc., CA, USA) and were analyzed through tumor Scan (TuScan) and BioDiscovery’s SNP-FASST2 algorithm using Nexus Express for OncoScan software version 7.5 [51 (link)].

For in vitro samples, purified B1-8^hi B cells were set in culture with purified OT-2 T cells and stimulated for 3 days with 1 µm beads coated with NP-OVA (1:3 B cell:beads ratio) or 25 µg/mL LPS. For in vivo samples, mice were immunized i.p. with 200 µg NP-CGG complexed with alum for 7 days. Cells were recollected and spleens were harvested. Single-cell suspensions were stained for a live/death marker, anti-CD19, -GL7, and -CD95, as explained previously. GC B cells were sorted (CD19 + GL7 + CD95 + live/death−). Naive follicular B cells were sorted based on the expression of CD19, CD23, and CD21. RNA from sorted cells was purified using an RNAeasy Plus Mini kit (QIAGEN). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent). Labeling and hybridizations were performed according to protocols from Affymetrix. Briefly, 100 ng of total RNA were amplified and labeled using the WT Plus reagent kit (Affymetrix) and then hybridized to Mouse Gene 2.0 ST Array (Affymetrix). Washing and scanning were performed using an Affymetrix GeneChip System (GeneChip Hybridization Oven 645, GeneChip Fluidics Station 450, and GeneChip Scanner 7 G). Gene Set Enrichment Analyses were performed using GSEA v2.2.2.

The integrity of total RNA extracted from spermatogonia and spermatocytes of CL (n=3), YK (n=3) and CY (n=3) was evaluated by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US) and up to the requirement of Affymetrix miRNA 4.0. The RNA labeled with FlashTag Biotin HSR was stained after hybridization and the slides were immediately scanned using GeneChip® Scanner 7G (Affymetrix, Santa Clara, CA, US). Command Console Software 3.2 (Affymetrix, Santa Clara, CA, US) was used to analyze array images to get raw data and then Expression Console (Affymetrix, Santa Clara, CA, US) offered RMA+DABG (Robust Multi-array Average plus Detection Above the Background) normalization. To define the differential expression profiles within the different variants, a one-way Anova was performed in the SAS software. The significant DE miRNAs were selected according to |log₂ (Fold change)| ≥ 1 and P-value < 0.05.