Total RNA was extracted from Paxgene-cryopreserved whole blood using PreAnalytiX standard protocol. Quality was verified using Agilent Bioanalyzer TapeStation
Label
biotin
Label protocol
The quality of total RNA was first assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Biotin-labeled cDNA targets were synthesized starting from 150 ng of total RNA. Double stranded cDNA synthesis and related cRNA was performed with GeneChip® WT Plus Kit (Affymetrix, Santa Clara, CA). With the same kit was synthesized the sense strand cDNA before to be fragmented and labeled. All steps of the labeling protocol were performed as suggested by Affymetrix, starting from 5.5 ug of ssDNA. Each eukaryotic GeneChip® probe array contains probe sets for several B. subtilis genes that are absent in the samples analyzed (lys, phe, thr, and dap). This Poly-A RNA Control Kit contains in vitro synthesized, polyadenylated transcripts for these B. subtilis genes that are pre-mixed at staggered concentrations to allow GeneChip® probe array users to assess the overall success of the assay. Poly-A RNA Controls final concentration in each target are lys 1:100,000, phe 1:50,000, thr 1:25,000 and dap 1:6,667.
Hybridization protocol
Hybridization was performed using the GeneChip® Hybridization, Wash and Stain Kit. It contains mix for target dilution, DMSO at a final concentration of 7% and pre- mixed biotin-labelled control oligo B2 and bioB, bioC, bioD and cre controls (Affymetrix cat #900299) at a final concentration of 50 pM, 1.5 pM, 5 pM, 25 pM and 100 pM, respectively. Fragmented and labeled sscDNA were diluted in hybridization buffer at a concentration of 23 ng/ul for a 2.3 ug total and denatured at 99 °C for 5 minutes incubated at 45 °C for 5 minutes and centrifuged at maximum speed for 1 minute prior to introduction into the GeneChip® cartridge. A single GeneChip® Human Clariom S was then hybridized with each biotin-labeled sense target. Hybridizations were performed for 16 h at 45 °C in a rotisserie oven. GeneChip® cartridges were washed and stained with GeneChip® Hybridization, Wash and Stain Kit in the Affymetrix Fluidics Station 450 following the FS450_0007 standard protocol, including the following steps: (1) (wash) 10 cycles of 2 mixes/cycle with Wash Buffer A at 30 °C; (2) (wash) 6 cycles of 15 mixes/cycle with Wash Buffer B at 50 °C; (3) stain of the probe array for 5 min in SAPE solution at 35 °C; (4) (wash) 10 cycles of 4 mixes/cycle with Wash Buffer A at 30 °C; (5) stain of the probe array for 5 min in antibody solution at 35 °C; (6) stain of the probe array for 5 min in SAPE solution at 35 °C; (7) (final wash) 15 cycles of 4 mixes/cycle with Wash Buffer A at 35 °C; (8) fill the probe array with Array Holding buffer.
Scan protocol
GeneChip arrays were scanned using an Affymetrix GeneChip® Scanner 3000 7G using default parameters. Affymetrix GeneChip® Command Console software (AGCC) was used to acquire GeneChip® images and generate .DAT and .CEL files, which were used for subsequent analysis.
Description
PBMC from CD patient at baseline, Responder at week 14
Data processing
Preprocessing was perfomed in R using the oligo package. Raw CEL files were summarized and normalized using RMA and quantile normalization (oligo::rma), using the probe definition and annotation packages pd.clariom.s.human and clariomshumantranscriptcluster.db
