Staining of Dissociated Lung Cells for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE sequencing)

Frozen cells were thawed in 37°C water bath and soon after the freezing medium melted cells were transferred into a 15 ml conical tube and the tubes were filled with staining buffer [1% BSA (w/v, Millipore) in DPBS (Biowhittaker)] to dilute the DMSO in freezing medium.

Cells were centrifuged at 800g, 4°C for 10 minutes.

After centrifugation supernatants were discarded and cells were resuspended in 1.0 ml staining buffer.

Viable cell counts were determined by trypan blue exclusion assay

Cells were then incubated in staining buffer containing 2% v/v normal mouse serum (Millipore Sigma) for 10 minutes over ice for Fc-receptor blocking.

Cells were then washed with staining buffer and incubated at a final concentration of 1 × 10⁶ cells per 100 μl of staining cocktail containing analyte-specific antibodies ( attached table ) for 30 min at 4°C.Covid Supp CITE Seq Staining Antibody List Gautam B.xlsx

After antibody-staining, cells were washed three times (800g, 4°C for 10 minutes) and resuspended in 100 μl staining buffer and passed through a 100-μm strainer (Falcon, Corning) and sent Genomic Research Center, University of Rochester Medical Center for further processing for CITE sequencing.