title: GSM5916734: Infection2_D8_SEQ1; Plasmodium vivax; Homo sapiens; Mus musculus; RNA-Seq
library_construction_protocol: Briefly, MPCCs were first treated with AP20187 (0.5 mM) for 30 minutes at 37°C to partially deplete the fibroblast cells via apoptosis. After washing, the cultures were dissociated by Trypsin (0.25%) 5-minute treatment at 37°C. For the scRNA-seq analysis, an updated Seq-Well protocol including a second-strand synthesis step was employed (Hughes et al., 2020). A suspension with 10-15,000 cells (pooled from duplicate MPCC wells) was then loaded onto a functionalized-polydimethylsiloxane array preloaded with uniquely barcoded mRNA capture beads. For each time-point in the two independent infections, two separate arrays were loaded with P. vivax-infected samples, and single arrays for mock and naïve samples. After cells had settled into wells, the array was sealed with a hydroxylated polycarbonate membrane with a pore size of 10 nm, facilitating buffer exchange while permitting cell lysis, mRNA transcript hybridization to beads, and bead removal before proceeding with reverse transcription. The obtained bead-bound cDNA product then underwent Exonuclease I treatment to remove excess primer before proceeding with second-strand synthesis and PCR amplification. To capture parasite reads from Seq-Well, full length cDNAs were amplified an additional 5 cycles using Kapa HiFi polymerase including a 3-minute extension time to increase concentration for capture. 200-300 ng of cDNA was concentrated using a speedvac, reconstituted in 3.4 L water and hybridized for 32 hours as previously described (Gural et al., 2018). 10 L of capture material was amplified for 15 cycles following the standard protocol. Captured cDNA was then prepared into Illumina libraries using NexteraXT (Illumina). Final libraries were quality controlled using Fragment Analyzer (Agilent) and qPCR prior to Illumina sequencing (NextSeq500). Sequencing of captured parasite transcripts was performed for the two infection batches.