Blood and cell culture kit

Hybrid clones originated from an experimental cross of T. b. brucei J10 and 1738 [2 (link)] and comprised two diploid progeny F1R1 and F1G2 (originally called SG22 clone 16 and SG22 clone 7 respectively), which were subsequently used in F1 crosses [41 (link)], and two hybrid progeny with raised DNA contents presumed to be triploid (F1R3N = SG1 clone 18) or tetraploid (F1Y4N = SG1 clone 4; Table 1). The four hybrid clones were grown as procyclics in Cunningham’s medium (CM) [74 (link)] supplemented with 10 μg/ml gentamycin, 5 μg/ml hemin and 15% v/v heat-inactivated foetal calf serum (FCS) at 27°C. High molecular weight DNA for genome sequencing was purified from approximately 5 x 10⁸ trypanosomes using a Blood and cell culture kit (Qiagen) and a modification of the manufacturer’s yeast cell protocol. Briefly, cells were pelleted by centrifugation, washed once with PBS and resuspended in 5 ml lysis buffer containing proteinase and RNAase as per the manufacturer’s protocol. Following 1 hour incubation at 50°C, lysates were centrifuged at 5000 rpm for 5 minutes at room temperature in a microfuge to pellet debris before the supernatant was applied to a Genomic-tip 100/G column (Qiagen). Subsequent processing followed the manufacturer’s protocol; after isopropanol precipitation, DNA was resuspended in 200 μl 10 mM Tris, 0.1 mM EDTA, pH 8 and stored at 4°C.

Freshly isolated Cas9⁺c-Kit⁺dsRed⁺ leukemia cells were transduced with the lentiviral CRISPR library by spinoculation as described above. Twenty-four hours post transduction, the cells were transplanted into sublethally irradiated (600 cGy) recipient mice. The mice were sacrificed on day 12, and the bone marrow cells were harvested. Genomic DNA was isolated from the cells collected at 24 hours (T₀) post transduction and on day 12 (T₁₂) post transplantation using a Blood and Cell Culture Kit (QIAGEN). For each sample, a minimum of 4.5 μg (corresponding to on average ~500 cells per sgRNA) of genomic DNA was used for PCR amplification, and the representation of sgRNAs was assessed by next-generation sequencing (NGS) as described above. The raw reads were then normalized in Excel (Microsoft), and the in vivo fold-change of the sgRNAs was calculated by dividing the representation of each sgRNA at T₁₂ versus T_0. The sgRNAs were ranked based on the median fold-change of the biological replicates.