Single cell 5 library and gel bead kit v1

CIART^−/− and wild-type hPSC-AWOs were infected with SARS-CoV-2 (m.o.i. = 0.1). The hPSC-AWOs were dissociated into a single-cell suspension using Accutase cell detachment solution (Innovative Cell Technologies) at 24 h.p.i. and incubated at 37 °C for approximately 5 min, followed by gentle pipetting to break apart groups of cells. The cells were then washed twice in 1×PBS and filtered using a 40-μm Flowmi cell strainer (Bel-Art Scienceware). Cell counts and viability were then determined using trypan blue staining and a Countess II automated cell counter (Thermo Fisher Scientific). Target cell inputs of 10,000 cells for each condition were then loaded into a Chromium Controller using Chromium Next GEM (Gel Bead-In Emulsion) single cell 5′ library and gel bead kit v1.1 (10x Genomics) according to the manufacturer’s instructions. After the generation of GEMs, cDNA synthesis and library preparation of all samples was completed using the Chromium single cell 5′ library kit v1.1 (10x Genomics) according to the manufacturer’s instructions.

Using a Single-Cell 5′ Library and Gel Bead kit v.1.1 (10X Genomics, 1000165) and Next GEM Chip G Single-Cell kit (10X Genomics, 1000120), the magnetically enriched, FACS-sorted BMPC single cell suspension was loaded onto a Chromium single-cell controller (10X Genomics) to generate single-cell gel beads in the emulsion (GEMs) according to the manufacturer’s protocol. 4,000 – 15,000 cells were added to each channel and approximately 50 – 70 % were recovered (Supplementary Table 2). Captured cells were lysed and released RNA was barcoded through reverse transcription in individual GEMs. The 5′ gene expression (GEX) libraries, single-cell V(D)J libraries (1000016) and cell surface protein libraries were constructed according to manufacturer protocols. Library quality was assessed using a 2200 TapeStation (Agilent). mRNA libraries were sequenced to an average of 30,000 reads per cell using Illumina Novaseq sequencer with a paired-end 150-bp (PE150) reading strategy (Dana Farber Cancer Institute).