Magnetic activated cell sorting columns

LSK cells were sorted from FLs of 14.5-dpc embryos after c-Kit (CD117) enrichment using magnetic-activated cell-sorting columns (Miltenyi Biotec). 10,000 LSK cells were simultaneously transduced with mouse stem cell virus (MSCV)–Meis1a-puro and MSCV–Hoxa9-neo retroviruses and subsequently subjected to three rounds of CFC assays in MethoCult (M3231) supplemented with 20 ng/ml stem cell factor, 10 ng/ml IL-3, 10 ng/ml IL-6, and 10 ng/ml granulocyte/macrophage stem cell factor. Colonies were counted 6–7 d after plating, and 2,500 cells were replated. Similarly, 200,000 FL c-Kit⁺ cells were transduced with MSCV–AML1-ETO9a-neo, MSCV–MLL-AF9-neo, or MSCV–MLL-ENL-neo and subsequently plated into methylcellulose.

RNA-Seq data were obtained from CD34⁺ cells isolated from bone marrow samples of eight MDS patients (four RARS and four RCMD-RS) with SF3B1 mutation (four K700E, one E622D, one R625L, one H662Q and one K666R; 45–52% SF3B1-mutant allele expression range), four MDS cases (all refractory cytopenia with multilineage dysplasia) without mutations in the splicing factor genes SF3B1, SRSF2, U2AF1 and ZRSR2, and five healthy individuals.^{20 (link)} CD34⁺ cells were isolated from bone marrow samples of the 12 MDS patients and five healthy controls using magnetic-activated cell sorting columns (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's recommendations.
DNase-treated (Invitrogen, Carlsbad, CA, USA) total RNA was purified using XP beads (Beckman Coulter, High Wycombe, UK), and library preparation was performed using the NEBNext Ultra directional RNA Library prep kit (NEB, Hitchin, UK) following the manufacturer's recommendations. Custom indexes were used and samples were purified using XP beads (Beckman Coulter) instead of size selection. Sequencing was performed on an Illumina HiSeq2000 instrument (Illumina, San Diego, CA, USA).

Adapted from the work of Boyle et al. and Wilson et al. (Boyle et al., 2010b (link); Wilson et al., 2013 (link); Wilson et al., 2015 (link)) Briefly, 3D7_Hyp1-Nluc schizonts (~38-44 hpi) were concentrated using magnetic-activated cell sorting columns (Miltenyi Biotec) and incubated with the cysteine protease inhibitor E64 (10 µM) for ~4-6 h until discrete merozoites were visible inside the majority of schizonts. Approximately 30 min prior to the completion of this incubation period, test compounds and uninfected RBCs were dispensed into triplicate wells of a 96-well U-bottom plate. Schizonts were then mechanically ruptured by passage through a 1.2 µm syringe filter and the released merozoites were immediately added to all wells to achieve a final haematocrit of 1%. PfATP4 inhibitors and chloroquine were administered at the concentrations used in the ring-stage growth inhibition assay and heparin was administered at 100 µg/mL (Boyle et al., 2010a (link); Dans et al., 2020 (link)). 0.032% (v/v) DMSO was included as a vehicle control. The plate was then agitated at 300 rpm at 37°C for 30 min to allow merozoites to invade RBCs. Pellets were washed three times, resuspended in drug-free supplemented RMPI medium and incubated at 37°C for 24 h. Parasite biomass was then quantified as in the nanoluciferase egress and invasion assay and normalised to the vehicle control.

Plasma was obtained following centrifugation of blood ethylenediaminetetraacetic acid-collection tubes. Peripheral blood mononuclear cells (PBMCs) were separated by gradient centrifugation in cell preparation tubes. CD4⁺ T cells were isolated from PBMCs using a CD4⁺ T-cell negative isolation kit and magnetic-activated cell sorting columns (Miltenyi Biotec, Teterow, Germany; purity >95%). One million CD4⁺ T cells were lysed in QIAgen lysis (RLT) buffer, and the lysates were stored at −80 °C until the RNA and DNA were extracted using the QIAgen AllPrep DNA/RNA Mini Kit (Cat.no. 80204, QIAgen, Melbourne, Australia). During RNA purification, RNA columns were treated twice with QIAgen RNase-Free DNase. Plasma HIV-1 RNA for sequencing was extracted from 2 to 3 ml of plasma by ultracentrifugation and a guanidinium-based method, including a prespin to sediment potential cell remnants [10 (link)]. HIV-1 RNA was extracted from 10 to 100 μl VOA supernatant using the same method as the plasma samples, but without the prespin. From cell-associated HIV-1 RNA, plasma HIV-1 RNA and VOA HIV-1 RNA, we generated complementary DNA using the Superscript III (Invitrogen by Thermo Fisher Scientific) cDNA synthesis kit and a gene-specific primer for p24-RT according to the manufacturer's instructions. See Supplemental Digital Content 2 for primers and PCR conditions.