Nucleospin mini kit

The indoor and outdoor aerosol in PBS (K-O and K-I) was concentrated by adding 12% beef extract (Sigma, Oslo, Norway) and centrifugation at 10,000× g for 30 min. The pellet was treated with 600 µL of lysis buffer (RAV1) at 70 °C for 5 min from the NucleoSpin Mini kit (MachereyNagel, Cedex, France). The lysate was subsequently purified and washed following the manufacturer’s instructions. The Anderson discs collected from Farwaniya Hospital were swabbed several times with sterile PBS to collect the microbial load. The swabs were then processed similarly to the above-mentioned protocol. Aerosols were processed for RNA isolation from HI-1and HI-2 through the standard Trizol^® procedure [21 (link)]. Nucleic acid quantification was performed on a Qubit fluorometer employing the Qubit HS DNA/RNA assay kits (Thermo Scientific, Waltham, MA, USA). All the RNA samples (HI-1 and HI-2), were converted to complementary DNA (cDNA) before conducting the PCR [20 (link),21 (link)]. K-O and K-I samples collected in a month (n = 4; one sample per week) were pooled due to their low biological yields.

RNAit tool was used to design RNAi target against PEX1 coding sequence (CDS), the selected RNAi target has no significant similarity to other CDSs in T. brucei. PEX1 RNAi construct was stably transfected into bloodstream form trypanosomes and following clonal selection, the cells were seeded at a density of 0.5 million cells/ml and treated with DMSO as negative control (-Tet) or RNAi-induced with 2 µg/mL tetracycline (+Tet) in biological triplicates. The growth of the cells was monitored daily by cell counting using the Neubauer chamber over a period of 6 days, both in the presence (+Tet) and absence of inducer (-Tet i.e., equivalent DMSO). Cells were harvested on days 1 and 2, and RNA was isolated using NucleoSpin^® Mini kit (Macherey Nagel). Quantitative Realtime PCR (qRT-PCR) was performed using GoTaq^® 1-Step RT-qPCR kit (Promega) with primers specific for PEX1 (RE7039-RE7040) and Tubulin (control, RE7000-RE7001), respectively. qRT-PCR of the samples was performed using Rotor-Gene™ 6000 (Qiagen). The results were analyzed using double delta Ct method (Livak and Schmittgen, 2001 (link)) and graphically plotted using GraphPad Prism 10 software. To study the effect of proteasomal inhibition, cells were treated with 25 µM MG-132 (Sigma) for 6 h, along with tetracycline induction at 2 µg/mL.

cDNA of H1581 cells (100 ng) was used to amplify FGFR1 by attB-overhang primers and flip it into pDONR.221 using the BP-clonase (Invitrogen, Thermo Fisher Scientific). Bacterial transformation of the competent E. coli strain DH5α (Invitrogen, Thermo Fisher Scientific) was carried out according to the manufacturer’s instructions. Single clones were sequenced from mini-preparation of plasmid DNA using the NucleoSpin Mini Kit (Machery Nagel). For midi-preparation of plasmid DNA we used the NucleoBond Xtra Midi EF Kit (Machery Nagel). The different FGFR1 variants were generated by side using Gibson Assembly and the primers 234_F_ΔEC-21-FGFR1, 235_F_ΔEC-30-FGFR1, 236_F_ΔEC-85-FGFR1, 237_F_ΔEC-144-FGFR1, and 211_R_FGFR1_GA (Supplemental Table 1).

Ten recombinant clones resistant to tobramycin and susceptible to chloramphenicol were recovered from faeces of mice colonized with MG/intI1 strain and treated with ciprofloxacin. Plasmid extractions were performed using the NucleoSpin mini kit (Macherey Nagel) and pZE1intI1 plasmid was further isolated. The region encompassing the PintI1 promoter to the end of intI1 gene was first amplified by PCR using primers ApXFP3 and pZE12rev (S2 Table) with the PrimeSTAR Max DNA Polymerase (Takara) following the manufacturer’s instructions. PCR products were analysed by Sanger sequencing with primers ApXFP3, Int1LC18 and pZE12rev (S2 Table).

Sec16A-depleted cells, Sec16B-depleted cells or control cells grown in RPMI medium or incubated in KRBm for 4 h were spun down (4 min at 200 g) and washed in PBS prior to RNA extraction. For each condition, RNA was extracted using the NucleoSpin Mini Kit (Macherey Nagel, 740955.50). The RNA concentrations were measured using a NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Scientific). 1 μg RNA was used to synthesize cDNA using the GoScript Reverse Transcription System kit (Promega, A5000).
For each condition, a PCR was performed using Taq polymerase (Promega, M7841) and visualized on agarose gel to assess Sec16A and Sec16B. PCR primers used to detect Sec16A and Sec16B are listed in Table S2.