GSM8291121: Shrimp allergic patient, with tropomyosin stimulation, peripheral blood mononuclear cells; Homo sapiens; RNA-Seq
Collect PBMCs and serum from the patient. Centrifuge the solution down at 1100g for 10 minutes. Then the blood was specifically isiolated from the tube and transferred to a new tube. Centrifugation at various speeds was performed again to isolate the PBMCs from the serum layer. The PBMCs were counted and viability was assessed using an automated counter. If the viability was >95%, the PBMCs were loaded at a concentration of 10,000 cells into the 10X Genomics 5' Sequencing Chromium Chip. Used the 10X Genomics 5v2 library construction kit. PBMCs from unstimulated and stimulated healthy control and shrimp allergic pateints were resuspended in the master mix and loaded with the commercial provider's partitioning oil and gel beads to generate the gel in bead emulsion (GEMs) using the 10X genomics Chromium Controller. The poly-A RNA were retrotranscribed to cDNA to incorporate the Illumina R1 sequence (read 1 sequencing primer), the 16 nt 10x Barcode, the 10 nt unique molecular identifier, and the 13 nt template switch oligo. The pooled barcoded cDNA was cleaned using the Silane DynaBeads, amplified with PCR, and the appropriately sized fragments were selected with the SPRIselect reagent for downstream library construction steps. During these library construction steps the P5, P7, i5 and i7 sample indices as well as the Illumina R2 sequence (read 2 primer sequence) were added.