Skills

NGS next generation high throughput sequencing, ribosome profiling, RNA-seq

• Illumina sequencing from cDNA libraries prepared with Lexogen RNA-seq prep kits and in-house, modified and optimized protocols developed for ribosome profiling.
• Cryptococcus neoformans in yeast form under drug treatment of sertraline, fluconazole and their combination.
• Cryptococcus neoformans mating samples for studying ribosomal proteins during sexual cycle.
• Neurospora crassa inositol biosynthetic gene regulation.
• Lab-optimized denaturing urea gel and native gel electrophoresis for size selection, identifying linker ligation, reverse transcription and circularization of nucleic acids. 
• Bioinformatics analysis using STAR alignment, DESeq2 for mapping and differential gene expression and R based tools for data visualization.

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RT-qPCR & PCR

• Polymerase chain reaction PCR with Taq polymerase or high fidelity enzymes such as Phusion polymerase, Vent polymerase for PCRs from genomic DNAs, plasmids or cloning vectors, restriction endonuclease digested DNA fragments and different types of templates with varying GC content
• Real time quantitative PCR or RT-qPCRs using Applied Biosystem instruments such as Quant Studio 6 Flex or ABI 7300 with SyBR green reagent mix assembled in-house.
• Primers designed using Integrated DNA technologies platform or Primer 3 software maintaining features such as strong annealing to template and minimal level of dimer or secondary structure predictions
• Data analysis with Delta delta Ct method for qPCRs
• Inverse PCRs on nicked templates for plasmid sequencing
• Generation of cDNA with reverse transcriptase PCR
• Statistical analysis of standard error testing

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Cloning

• Used Gibson assembly and Restriction endonuclease based cloning
• Designing vectors and plasmids using NCBI and fungiDB sequence databases and DNAStar software program to generate gene/vector maps
• Used bacterial chemical transformation and fungal electroporation and homologous recombination to develop cloning vectors and mutant strains
• Screening using antibiotic selection plates
• Developed in vivo luciferase reporter strains of Neurospora
• Generation of Hygromycin resistant Neurospora transformants 
• Generated a phosphorescent Neurospora auxotropic mutant 

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Extraction of DNA/RNA/proteins

• Extraction of genomic DNA, RNA and proteins from bacteria and Neurospora
• Cryptococcal RNA extraction using lyophilized cells and zirconium beads
• Absorbance measurements spectrophotometrically to determine concentration of nucleic acids using Nanodrop and Cary spectrometer instruments
• Picogreen assay for DNA and Ribogreen assay for RNA standard curve and concentration measurements using Perkin Elmer plate reader
• Bradford assay for protein concentrations
• Luciferase bioluminescence assays using Envision plate reader for continuous in vivo reporter signals
• RNA extraction using Trizol from homogenized mammalian tissue such as rat cardiac sample showing hormone induced hypertrophy.
• SDS PAGE, Western Blotting and polysome extraction & gradient centrifugation for identification of proteins related to translation.

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