S3 sorter

Cells were isolated from mes-LN and BM on day 28 or later after infection and on day 10 after transfer and B cells were purified with the MojoSort™ Mouse Pan B Cell Isolation Kit II (BioLegend). For cells after transfer Fc receptors were blocked with anti-mouse CD16/CD32 mAb and the cells stained at 4°C for 30 min for CD45.2 (Biolegend). Cells were sorted for tdTomato⁺ and tdTomato^- before transfer and CD45.2⁺ tdTomato⁺ or CD45.2⁺ Tomato^- cells after transfer using the S3 sorter (Bio-Rad). The cells were sorted into 1 ml FCS, pelleted and immediately lysed with RLT-buffer (RNeasy-Kit; Qiagen). The lysate was stored at -80°C until RNA isolation. RNA was isolated using the RNeasy micro kit (Qiagen). The samples were prepared for sequencing and analyzed according to the procedures described in (31 (link)).Samples were sequenced in an illumina MiSeq machine using the MiSeq Reagent Kit v3 (600-cycle).

Cells were sorted on a FACS ARIA III (BD Biosciences) using a 70 μm nozzle, into FACS tubes (Falcon) containing 1 ml 4 °C PBS (Gibco). Sulphadiazine-enriched gametocytes (Supplementary methods) were sorted using a S3 sorter (BioRad). The cells were injected IV into uninfected mice (Supplementary methods) or centrifuged at 400×g for 10 min and the cell pellet smeared onto a standard microscope slide. The slides were stained in Giemsa or May-Grünwald (0.25% (w/v) in methanol) (Sigma-Aldrich) for 5 min, washed in 1% PBS (Gibco) being washed in water. The slides were examined under a standard light microscope using a ×100 objective and immersion oil.

The G₁ and G_d cells were sorted from the WT culture (treated and stained with Sytox Green) using a Biorad S3 sorter. Sorted cells were collected and resuspended in 50mM Tris buffer (pH 7.5) and stained with 0.1mg/ml of calcofluor white for 20min at 30°C. Stained cell were washed twice with buffer before imaging. Cells subjected to glucan staining were fixed with 70% ethanol for one hour, washed with PBS buffer twice, and stained with 5mg/ml of Aniline Blue for 20min.

Human monocyte-derived macrophages (HMDMs) were infected with Erdman smyc’::mCherry reporter Mtb strain at MOI 2:1. At 2 dpi, experimental compounds were administered at 10 µM, INH at 67.5 ng/mL, and EMB at 5 µg/mL. Infected HMDMs were treated for 48 h prior to live sorting using a Biorad S3 sorter. 40,000 mCherry positive cells were sorted into 700 µL of Trizol. To remove host RNA, Trizol samples were centrifuged and ~75% (~500 µL) of Trizol supernatant was transferred to a new tube. An additional 400 µL of Trizol and 150 µL of zirconia beads were added to the tubes containing the bacterial pellet and 25% of the remaining Trizol. These tubes were placed on a beat beater for two cycles of 1 min each, with 2 min of rest on ice in between. After the bead beating, approximately 75% of host RNA in Trizol was added to be bacterial lysate tubes. Chloroform was added to Trizol tubes (200 µL of chloroform for 1 mL of Trizol) and tubes were mixed via shaking vigorously. Trizol:chloroform tubes were centrifuged for 15 min and the aqueous phase (~1 mL) was transferred to a fresh tube. Two microliters of glycoblue and 500 uL of isopropanol were added to precipitate host and bacterial RNA. RNA was washed in 75% cold ethanol and RNA was eluted in 12 µL of NCFW.