Illumina HiSeq 3000 paired end sequencing; Comparative proteomic and RNA-seq analyses identify glypican-3 as a hepatocyte host cell receptor for Plasmodium falciparum sporozoite invasion
Comparative proteomic and RNA-seq analyses identify glypican-3 as a hepatocyte host cell receptor for Plasmodium falciparum sporozoite invasion
Cells were grown in appropriate media, in T75 flasks for 24 hours, media was removed and then cells were washed twice with PBS. TRIzol was added directly to the cells. Cells were split at a 90% confluent culture by digesting the monolayer in 5mL of 0.05% Trypsin-EDTA for 10 minutes or until cells lifted. The cell suspension was collected in a conical tube and centrifuged at 700xg for 7 minutes to ensure pelleting of the cells. Trypsin was removed, and cells were resuspended in media for plating, then plated at a 1:10 dilution in a new T75 flask in fresh IMDM with 5% HIFBS, 200 units/mL penicillin, and 200 µg/mL streptomycin for maintenance of line. Following addition of TRIzol, samples were transferred to 1.5 mL tubes. Preparation of RNA was done following manufacturers instructions. Approximately 500 ng of protein-free and intact total RNA was used for library construction using the reagents provided in the NEBNext Ultra II RNA Library Prep following the manufacturer's protocol. First, 2 µL of diluted RNA was spiked with ERCC from the kit. Next, mRNA isolation was performed using the NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs). Then RNA was fragmented in a solution containing divalent cations, with incubation at 94°C. Next, first strand cDNA synthesis using reverse transcriptase and random primers was done. Synthesis of double stranded cDNA was done using the second strand master mix provided in the kit, followed by end-repair and dA-tailing. At this point, Illumina adaptors were ligated to the sample. Finally, the library was enriched by 11 cycles of amplification and purified by Agencourt AMPure beads (Beckman Coulter). Barcoded libraries were sized on the bioanalyzer and quantitated by QUBIT. Quantitative PCR was used to validate the library's functionality, using the KAPA Library Quantification kit (Kapa Biosystem) and monitoring with the BioRad Touch Real-Time PCR Detection System. Individual libraries were pooled equimolarly for sequencing runs.
Experimental Factor: cell line
HC-04
Experimental Factor: growth condition
IMDM