Novaseq sp flow cell

Cells from HS360 hESCs were differentiated in one time-course experiment, and at Day 0 and Day 20, the cells were washed twice with 1xPBS and detached to single cell suspension by application of Accutase (STEMCELL Technologies) at 37°C for 7 min. The detached cells were washed with appropriate base media with added 0.04% BSA (Sigma-Aldrich) and filtered using MACS SmartStrainers (Miltenyi Biotech) to remove cell aggregates. Nuclei isolation was done according to the 10x Genomics protocol CG000169 (Rev D) using 2 min of incubation in lysis buffer diluted to 0.1x and 0.5x for Day 0 and Day 20 cells, respectively. We used the Countess II FL Cell Counter (Thermo Fisher Scientific) to quantify nuclei and confirm complete lysis and microscopy to confirm high nuclei quality. Nuclei were further processed on the 10x Chromium controller (10x Genomics) using Next GEM Chip H Single Cell Kit (10x Genomics), Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1 (10 x Genomics) and Chromium i7 Multiplex Kit N Set A (10x Genomics) according to the Next GEM Single Cell ATAC Reagent Kits v1.1 User Guide (CG000209, Rev C). The targeted nuclei recovery was 5,000 nuclei per sample. The resulting 4 sample libraries were sequenced on a NovaSeq Sp flow cell (Illumina) with 50 cycles for read 1, 8 cycles for the i7 index read, 16 cycles for the i5 index read and 49 cycles for read 2.

Cells were washed twice with 1xPBS and detached to single cell suspension by application of Accutase (STEMCELL Technologies) at 37°C for 7 min. The detached cells were washed with appropriate base media with added 0.04% BSA (Sigma-Aldrich) and filtered using MACS SmartStrainers (Miltenyi Biotech) to remove cell aggregates. Nuclei isolation was done according to the 10x Genomics protocol CG000169 (Rev D) using 2 minutes of incubation in a lysis buffer diluted to 0.1x and 0.5x for Day 0 and Day 20 cells, respectively. Countess II FL Cell Counter (ThermoFisher Scientific) was used to quantify nuclei and confirm complete lysis and microscopy to confirm high nuclei quality. Nuclei were further processed on the 10x Chromium controller (10x Genomics) using Next GEM Chip H Single Cell Kit (10x Genomics), Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1 (10 x Genomics) and Chromium i7 Multiplex Kit N Set A (10x Genomics) according to the Next GEM Single Cell ATAC Reagent Kits v1.1 User Guide (CG000209, Rev C). The targeted nuclei recovery was 5,000 nuclei per sample. The resulting 4 sample libraries were sequenced on a NovaSeq Sp flow cell (Illumina) with 50 cycles for read 1, 8 cycles for the i7 index read, 16 cycles for the i5 index read and 49 cycles for read 2.

The processing of spatial transcriptomics followed the commercial brochure of the Visium Gene Expression Kits (catalog no. PN-1000184, 10X Genomics). Briefly, freshly isolated ILNs and MLNs were frozen in 2-methylbutane (catalog no. M32631, Sigma-Aldrich) with liquid nitrogen and immediately embedded into frozen section media (FSC22, catalog no. 3801480, Leica Biosystems) for spatial transcriptomics. The tissue blocks were cut into 10-μm sections using a cryostat and mounted on Visium Gene Expression slides (catalog no. PN-1000188, 10X Genomics). Sections on Visium Gene Expression slides were first fixed with precooled methanol for 30 min and stained with 4′,6-diamidino-2-phenylindole, B220 and ACTA2 and imaged with confocal microscopy to visualize the fiducial frames of capture areas and cell compartmentalization in different sections. The optimal permeabilization time for in situ hybridization and reverse transcription of murine LN transcripts was 15 min evaluated with the 10X Genomics Visium Tissue Optimization Kit (catalog no. PN-1000193, 10X Genomics). cDNA libraries were generated using the Visium Spatial Gene Expression 3′ Library Construction Kit (catalog no. PN-1000190, 10X Genomics) and were sequenced on an Illumina NovaSeq SP flowcell by the Functional Genomic Center in Zurich.

Infection samples for mRNA sequencing were prepared by mixing ~10,000 bleach-synchronized L1 stage animals with 1 mL of 20X OP50-1 and 10 million N. parisii spores, 10 million N. ausubeli spores, 10 million N. ironsii spores, and 30 million N. ferruginous spores in four separate microcentrifuge tubes. Samples were then spread onto individual 10-cm NGM plates and allowed to dry before incubating at 21°C for 48 hours. Samples for RNA extraction were washed in M9/0.1% Tween-20 in 15 mL conical tubes at least three times before adding 1 mL of TRI-Reagent (Sigma-Aldrich). Total RNA extraction and ethanol precipitation protocols was performed as per TRI-Reagent manufacturers instruction. Libraries were prepared using NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina kit as per manufacturer’s instruction. RNA sequencing was carried out using Illumina NovaSeq-SP flow cell by The Centre for Applied Genomics (TCAG).

Libraries were prepared using a SciClone G3 NGSx workstation (Perkin Elmer) using the Kapa mRNA HyperPrep kit (Roche Sequencing). Polyadenylated mRNAs were captured using oligo‐dT‐conjugated magnetic beads (Kapa mRNA HyperPrep kit, Roche Sequencing) from 300 ng of total RNA on a Perkin Elmer SciClone G3 NGSx automated workstation. Polyadenylated mRNA samples were immediately fragmented to 200–300 bp using heat and magnesium. First‐strand synthesis was completed using random priming followed by second‐strand synthesis and A tailing. dUTP was incorporated into the second strand to allow strand‐specific sequencing of the library. Libraries were enriched and indexed using 12 cycles of amplification (Kapa mRNA HyperPrep kit, Roche Sequencing) with PCR primers, which included dual 8bp index sequences to allow for multiplexing (IDT for Illumina unique dual 8bp indexes). Excess PCR reagents were removed through magnetic bead‐based cleanup using Kapa Pure magnetic beads on a SciClone G3 NGSx workstation (Perkin Elmer). Resulting libraries were assessed using a 4200 TapeStation (Agilent Technologies) and quantified by QPCR (Roche Sequencing). Libraries were pooled and sequenced on one Illumina NovaSeq SP flow cell using paired‐end, 75 bp reads.