title: GSM8734763: Mediastinal lymph node biol rep 1, GEX; Mus musculus; RNA-Seq
library_construction_protocol: The lung and mediastinal lymph node were dissected from the same mouse (i.e. lymph node 1 is paired with lung 1, lymph node 3 is paired with lung 3). Lymph node samples were pressed through a wet 70 µm cell strainer, before being washed through with 3 mL of flow buffer (PBS + 2% FBS + 1 mM EDTA). Lungs were chopped using scissors before being digested for 30 minutes at 37°C in a 500RPM shaker, in RPMI containing 100 µg/mL Liberase TL (Roche, Basel, Switzerland) and 100 µg/mL DNase I (Roche). Lung pieces were then pressed through a wet 70 µm cell strainer. Lung samples were resuspended in ACK red cell lysis buffer, incubated for 5 minutes and then washed and resuspended in flow buffer. Cells were washed and resuspended in a fluorescently-conjugated antibody cocktail made up in flow buffer and stained for 1 hour. Live isotype-switched endogenous germinal centre B cells (CD45.2+B220+CD19+IgD-CD95+CD38-) and memory B cells (CD45.2+B220+CD19+IgD-CD95-CD38+) were sorted using a FACSAria Fusion (Becton Dickinson). Cells were sorted into flow buffer and prepared for 10x sequencing. Library was performed according to the manufacturer's instructions (single cell 5' v2 protocol, 10x Genomics). Briefly, FACS sorted cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identified (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.