Nanodrop analysis

Peripheral blood samples were collected from 14 AML patients with informed consents at Queen Mary Hospital, Hong Kong. Total peripheral blood mononuclear cells were purified according to a previous protocol.²¹ The samples were confirmed to have at least 38% of leukaemia blasts. Total RNA extraction was carried out using TRIzol (ThermoFisher) based on the manufacturer's instructions. The quality and quantity of RNA samples were evaluated by NanoDrop analysis (ThermoFisher) and agarose gel electrophoresis. RNA samples were reverse transcribed into cDNAs using a reverse transcription kit (ThermoFisher) according to the manufacturer's protocol. Taqman® miRNA assays (ThermoFisher) were used to quantify the levels of miR‐125a and miR‐125b, and U6b RNA was used as the internal control. Ssofast Green qPCR kit (Bio‐Rad) was used to quantify the levels of FLT3‐ITD mRNAs, relative to the level of GAPDH. VIIA(TM) 7 System (Applied Biosystems, USA) was used to process all qPCR reactions.

To exclude the very unlikely possibility that the gerBAC genes had moved to another place in the chromosome, the Δbont Δpyr ΔgerBAC strain and its parent Δbont Δpyr were subjected to whole genome sequence analysis on a Illumina MiSeq sequencer. First, gDNA from both strains was isolated from overnight cultures using the GeneJET Genomic DNA purification kit (Thermo Scientific). DNA purity and concentration was assessed by Nanodrop analysis, gel electrophoresis and Qubit (Thermo Scientific) analysis. Paired-end libraries were constructed using the NEBNext Ultra gDNA library prep protocol with an average insert size of 240 bp, and analyzed on the Agilent BioAnalyzer (VIB nucleomics core, Belgium) resulting in on average 1.2 million reads per sample. Reads were analysed with Qiagen’s CLC Genomics Workbench version 8.5 (http://www.clcbio.com/), and subjected to standard quality control, read trimming and filtering (reads <15 nucleotides were discarded, quality score limit = 0.01, ambiguous nucleotides trim limit = 2), and read mapping to C. botulinum NCTC 11219 reference WGS with accession number JXMR00000000 (using parameters: mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.8, similarity fraction = 0.8). This resulted in an average 43-fold genome coverage for the parental strain and 51-fold coverage for the ΔgerBAC mutant.

Synthesis of cDNA was performed using the SuperScript II Reverse Transcriptase kit (Life Technologies, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, 500 ng DNase-treated RNA was diluted in DEPC-treated water supplemented with 500 ng oligo(dT) primers (Stratagene, San Diego, CA, USA) and 10 nmol dNTP mix (Fermentas, Waltham, MA, USA) up to 12 µL total volume and incubated at 65 °C for 5 min. To this, 4 µL 5× First Strand Buffer and 2 µL 0.1 M DTT were added and then incubated at 42 °C for 2 min. Subsequently, 1 µL SuperScript II Reverse Transcriptase was added and incubated at 42 °C for 50 min to synthesise the cDNA. The reaction was inactivated by incubation at 70 °C for 15 min. The cDNA concentration was measured by NanoDrop analysis (Thermo Fisher Scientific, Waltham, MA, USA). Sufficient cDNA purity was indicated by A260/280 ratios of ~1.8 and A260/ratios of ~2.0–2.2.