GSM7956894: CL6_LPS2_Bursa_S10, snRNAseq; Gallus gallus; RNA-Seq
After euthanizing the birds, spleens and bursae were aseptically removed, and flash frozen in liquid nitrogen then stored at -80 until shipment. The nuclei isolation methods performed were as described in 10X Genomics Isolation of a Single Cell Suspension (10X Genomics). In brief, approximately 50 mg of tissue was homogenized using a glass Dounce homogenizer with ten repeated plunging movements, the homogenate was then filtered first on a 70-micron then a 40-micron cell strainer (Fischer Scientific, Waltham, MA). The nuclei were subsequently washed twice in wash buffer of PBS and 1% BSA. Nuclei were manually counted by staining with trypan blue and a hemocytometer. The nuclei suspension was resuspended in resuspension buffer to a targeted 3,000 nuclei/sample. Single nuclei capture and cDNA preparation were performed with the 10X Genomics Chromium Single Cell 3' kit, version 2, and lipid encapsulation instrument (10X Genomics, Pleasanton, CA) according to the manufacturer's recommendations. Pooled libraries were barcoded by sample and sequenced (2x 150 bp length) on an Illumina HiSeq 6000 instrument (Illumina, Inc., San Diego, CA) to achieve 50,000 reads per nuclei. The individual tissue-specific sequenced Gel Bead-In Emulsion (GEM) libraries were each processed with the CellRanger v7.0.1 pipeline (10X Genomics), which performs demultiplexing, alignment, barcode processing, and sample aggregation to create a cellular barcode by genomic feature matrix. The chicken reference used for all single-nuclei read alignments was bGalGal1b or GRCg7b. First, we used the “cellranger mkgtf” command to filter out everything except protein-coding genes and long non-coding RNAs from the annotation, and then use the “cell ranger mkref” command to create the necessary indices. We aligned reads to this modified GRCg7b reference and generated count matrices for each sample individually using the command “cellranger count” with default options. Molecular barcodes that were most likely to be ambient signals and doublets were eliminated using the default settings of cellranger's “remove-background” v.0.2.0 function, and cell counts were calculated using cellranger default parameters