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Sample GSM5916720

Query DataSets for GSM5916720

Status

Public on Feb 25, 2022

Title

Infection2_D1_Uninfected

Sample type

SRA

Source name

day 1

Organisms

Homo sapiens; Mus musculus

Characteristics

cell type: primary human hepatocytes, with 3T3-J2s
infection: non-infected
treatment: non-treated

Treatment protocol

MPCCs were infected with fresh sporozoites obtained through hand dissection of P. vivax-infected mosquitoes. For mock samples, MPCCs were exposed to material from non-infected mosquito salivary glands (matched number of dissected mosquitoes and processed similarly as the infected counterparts). Naïve samples received the culture media used for the infections. Pi4K treatments were conducted from days 5 to 8 after infection.

Growth protocol

Primary human hepatocytes were seeded on collagen-micropatterned 96-well plates and surrounded with murine embryonic fibroblasts 3T3-J2s as detailed previously (March et al., 2015). For the scRNA-seq analysis, MPCCs were established using 3T3-J2s expressing an inducible apoptosis switch (Chen et al., 2020).

Extracted molecule

polyA RNA

Extraction 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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Two processed data files represent the sequencing of the library upon its construction (pre-capture), as well as a re-sequencing post-enrichment for parasite-derived transcript (post-capture data).
Precapture_Infection2.tar
Postcapture_merged_Infection2.tar

Data processing

For each sample, fastq files originating from multiple sequencing runs were concatenated and processed using an analytical pipeline derived from the DropSeq pipeline v. 1.12, as described in (Gierahn et al., 2017) (https://github.com/broadinstitute/Drop-seq).
Briefly, reads were converted to a bam file using picard v. 2.9.0-1-gf5b9f50-SNAPSHOT, tagged with cell and transcript barcodes, and subsequently sequencing adapters and polyadenosine tracts were trimmed. Upon regenerating fastq files, reads were aligned with STAR v. 2.5.3a (Dobin et al., 2013) against the aforementioned tri-genome reference. Genomic features of the aligned reads were annotated using the combined species-gene symbol nomenclature including gene and exon of origin when relevant. Bead synthesis errors were assessed and when possible, altered unique molecular identifiers (UMIs) were repaired. Cell barcode abundance was tallied, and gene expression was called for the top 10,000 cell barcodes.
Count matrices of genes x cells were imported in the R v. 3.6 statistical environment and Seurat v.3 was used as the primary analytical package (Satija et al., 2015). Count matrices were merged into a single Seurat object, which was split by the sample of origin. Human, murine and P. vivax genes and transcripts counts were tallied for each cell barcode.
Genome_build: Tri-genome hg19, mm10 (ENSEMBL release as used in Macosko et al. Cell 2015) and PvP01 v1.
Supplementary_files_format_and_content: Tab-delimited text files containing unfiltered genes x cell-barcode matrices in MatrixMarket format (10K cells called for each sample). Three files are reported: for "Experiment 2", a pre-parasite capture count matrix is provided (containing host and parasite data for 10K cells per sample), as well as a post-capture file, which contains data for 10K cells called upon sequencing the captured libraries, added to the corresponding reads tallied pre-capture for each matching cell barcode. For "Experiment 1" the post-capture data only (10K cells/sample).

Submission date

Feb 24, 2022

Last update date

Feb 25, 2022

Contact name

Liliana Mancio-Silva

E-mail(s)

liliana.mancio@pasteur.fr

Organization name

Institut Pasteur

Street address

25-28 Rue du Dr Roux