GSM6598833: SC333; Homo sapiens; RNA-Seq
Explanted subpleural peripheral lung tissue was finely diced and digested for 1 hour in a mix of either Collagenase A/DNase I or Liberase DL/DNase I.  A single cell suspension was generated by filtering lungs through 70 micron filters and resuspending cells in PBS containing 0.04% BSA.  For some lungs, CD45+ and EPCAM+ cell populations were partially depleted with Miltenyi magnetic bead separation.  For scATAC, cells were lysed according to 10X Genomics' Nuclei Isolation for Single Cell ATAC Sequencing protocol (CG000169 ).  Cells or nuclei were loaded into the 10X Genomics Chromium instrument per manufacturer's directions. Libraries were constructed per the manufacturer's instructions (Single Cell 3' v2, Single Cell 5' v1, scATACv1 protocols, 10x Genomics). In short, for the scRNA protocols, lung cells were added to the master mix and loaded into the chip; subsequently, gel beads and partitioning oil were added into the chip which was then loaded into the Chromium instrument, generating the gel bead-in-emulsion (GEM). Once emulsified, cells were lysed and poly-A RNA transcribed to cDNA, adding an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and 10x Barcode. Silane DynaBeads were then used to clean up the pooled and barcoded cDNA which was then amplified by PCR.  SPRIselect reagent was used to select the appropriately sized fragments for library construction. Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added during the library construction protocol.  For scATAC, nuclei were incubated for one hour with the transposase then loaded into the Chromium instrument with barcoding reagents, gel beads, and partitioning oil.  Once emulsified, the P5 sequence and 10X barcode were added during the PCR GEM generation and barcoding steps.  The resulting 10x barcoded single-stranded DNA was then cleaned up with Silane Dynabeads and size selected with SPRIselect reagent.  Finally, a P7 sequence and a sample index were added during library construction via PCR.