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Lung single cell suspension and CD45+ cell enrichment

The core has used CD45 enrichment to capture rare cells such as innate lymphoid cells (ILCs). In brief, after euthanasia, murine lung tissues were isolated and minced with forceps and small scissors and digested in 2mL IMDM medium without serum with 2 mg/mL collagenase (Sigma, C0130-1G) and 20U/mL DNase-1 (Sigma, DN25-100MG) for 90 min at 370C in cell culture incubator with gentle rotating. Digested tissues were passed through a sterile 70 mm filter (Fisher Scientific, #22363548) to obtain a single cell suspension. After washing, 2mL ACK Lysing Buffer (Thermo Fisher Scientific, A10492) was added into the cell suspension and incubated for 2 minutes at room temperature, and washed twice with complete IMDM additionally. Cell counts and cell viabilities were validated by Cellometer (Nexcelom Bioscience LLC, MA). When whole lung single cell suspensions were obtained. 1x106 cells per condition were saved to represent the whole lung single cell population. The remaining cells were then subjected to CD45 enrichment by applying Ly-6G negative selection (Miltenyi, #130-092-332) and followed by CD45 positive selection (Miltenyi, #130-052-301). Briefly, the cells were suspended in MACS separation buffer, incubated with the anti-Ly6G-biotin for 10min at 40C followed by anti-biotin microbeads for 15min at 40C, and applied to a MACS LS separation column. The flow through was collected as the Ly6G negative (Ly6G-) population. The Ly6G- cells were then incubated with anti-CD45 microbeads and applied to a MACS MS column. After washes, the CD45 positive (CD45+) population was released from the column by flushing the column and collected into an Eppendorf tube. Both whole lung single cell and CD45 enriched populations were treated with 100uL TrypLE for 1 minute to dissociate single cells from any small aggregates or clusters. Cell numbers and viabilities were then validated by Cellometer prior to single cell RNAseq library preparation.

Reagent:

Reagents used for MACS were purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany), and include: anti-Ly-6G (cat no. 130-092-332) and CD45 (cat no. 130-052- 301) MicroBeads.