title: GSM9382825: PFF Duodenal Immune Cells 1; Mus musculus; RNA-Seq
library_construction_protocol: Harvested duodenum was mechanically cut into fine pieces and then digested in Accumax for 5 minutes at 37 degrees C with vigorous shaking. The digested solution was then filtered, counted, and split in half for 2 parallel simultaneous separation protocols. One half was subject to CD45-FITC antibody and DAPI labelling followed by FACS-based enrichmen. The other half was subject to CD11b magnetic bead based enrichment followed by DAPI labelling with FACS-based exclusion of dead cells. The enriched fractions from each protocol were then combined before loading onto the 10x Chromium. Library construction was performed according to the 10x single-cell 3' v3 protocol. Briefly, the master mix was combined with the partitioning oil and sample then loaded onto the chip for creation of gel-bead emulsions (GEM). Each individual GEM reaction produced poly-A RNA that was converted to cDNA tagged with an Illumina R1 transcript, Unique Molecular Identifier (UMI) and 10x barcode. The cDNA library was cleaned with DynaBeads, amplified by PCR, and separated for size-appropriate fragments using SPRI-select. Quality-checking of the library was performed with Agilent Bioanalyzer. Library construction was completed by adding the paired-end fragments and indexing samples as approrpaite.