GSM7156477: Pooled mouse experiment; Mus musculus; RNA-Seq
Murine lungs were dissociated using a GentleMACS Dissociator (Miltenyi Biotec) in digestion buffer (RPMI with 10mM HEPES, DNAse I 50ug/ml, Liberase TM 100ug/ml, and 2% FBS). After running the m_lung_01 program, samples were incubated at 37C for 30 minutes before running the m_lung_02 program. Samples were filtered with a 70 uM filter, washed once, then RBCs were lysed for 5 minutes using RBC Lysis Buffer (Sigma-Aldrich). Samples were then quenched with FACS buffer (PBS with 2% FBS and 2 mM EDTA) and washed once. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Cells were then counted prior to proceeding with MULTI-seq barcoding. Samples were multiplexed as previously described. In brief, samples were barcoded with 2.5uM of the LMO anchor and barcode for 5 minutes on ice in PBS before adding 2.5 uM of the LMO co-anchor and incubating for an additional 5 minutes. Samples were quenched with 1% BSA in PBS and washed once. Barcoded 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 Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.