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Sample GSM2775681

Query DataSets for GSM2775681

Status

Public on Sep 08, 2017

Title

42hpi_D6

Sample type

SRA

Source name

Single cell sorted Plasmodium infected human erythrocytes

Organism

Plasmodium falciparum NF54

Characteristics

time point: 42hpi
induced: Induced

Treatment protocol

Parasite cultures were sorbitol-synchronized (Sigma-Aldrich) as described previously 21 to a 4-hour cycle window using 4 consecutive treatments across two growth cycles. At 28-32 hour post invasion, parasites were washed in RPMI/mFA media and plated with or without LysoPC. At 4, 8, and 12 hours post induction, cells were stained with Vybrant DyeCycle Violet (Life Technologies) in HBSS (Thermo Fisher Scientific). Stained cells were pelleted and resuspended in phenol red-free PBS supplemented with 0.3% RNaseOUT (Life Technologies). Collection Twin.tec PCR 384-well plates (Eppendorf) were prepared by addition of cell lysis buffer to each well; lysis buffer consisted of nuclease-free water (Thermo Fisher Scientific) supplemented with 0.2% HF buffer (New England Biolabs). Single Vybrant Dye Cycle Violet-positive cells were sorted using a Beckman Coulter MoFlo Astrios EQ. At each time point, 48 cells from the non-induced culture (+LysoPC) and 336 cells from the induced culture (-LysoPC) were sorted into the collection plate and snap frozen on dry ice. Cultures were maintained for an additional two cycles for parasitemia and gametocytemia measurement. The media of both non-induced and induced cultures was replaced with RPMI/serum 12 hours post induction and daily thereafter.

Growth protocol

P.falciparum cell culture was performed as described previously (REF). Culture media consisted of RPMI-1640 supplemented with 25 mM HEPES, 100 μM hypoxanthine, 24 mM sodium bicarbonate, and gentamicin (all from Sigma-Aldrich). Serum media was generated by additional supplementation of 10% O+ human serum (The Interstate Companies) while serum-free medium was generated by additional supplementation of 0.39% fatty acid-free BSA, 30 μM oleic acid, and 30 μM palmitic acid (all from Sigma-Aldrich). To generate serum-free media supplemented with 20 μM LysoPC, LysoPC (Avanti Polar Lipids) was dissolved in ethanol and dried on culture dishes prior to culture addition. All cell cultures were maintained in a 5% CO2/1 % O2 and 95 % N2 gas mixture (Med-Tech Gases). All experiments used a clone of the Pf2004/164TdTomato P.falciparum line 14, which requires addition of 4 nM WR99210 (Jacobus Pharmaceuticals).

Extracted molecule

polyA RNA

Extraction protocol

We used an optimized version of Single Cell RNA Barcoding and Sequencing 7 (SCRB-seq; Soumillon et al, 2014), further reducing the reverse transcriptase reaction volume. In brief, poly(A)+ mRNA from flow-sorted single Plasmodium infected erythrocytes were converted to cDNA, decorated with universal adapters, well barcodes, and unique molecular identifiers (UMIs), using a template-switching reverse transcriptase. Then, cDNA from multiple cells was pooled, amplified, and prepped for multiplexed sequencing using a transposon-based fragmentation method, enriching for 3’ ends and preserving strand information.
Nextera XT

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

GGACTCCT_D6
42hpi.raw.tsv
42hpi.normalized.tsv

Data processing

Illumina NextSeq standard basecalling platform
Well barcode demultiplexing. Custom python script. Separates wells in 384-well plate
Unique molecular identifier demultiplexing. Custom python script. Counts reads from same mRNA molecule as only 1
Transcript alignment with bwa aln v0.7.10 allowing 4 mismatches in seed sequence and rest of sequence.
Discard reads with polyA tails (> 20 As in a row)
Additional filtering: Filter reads for rRNA. Remove samples with fewer than 300 reads. Remove genes that were found in <20 samples.
Generate normalized abundances with edgeR v3.14.0
Genome_build: PlasmoDB Plasmodium falciparum 3D7 transcript v29
Supplementary_files_format_and_content: 38hpi.raw.tsv - 38hpi raw read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: 42hpi.raw.tsv - 42hpi raw read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: 46hpi.raw.tsv - 46hpi raw read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: 38hpi.filtered.tsv - 38hpi filtered read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: 42hpi.filtered.tsv - 42hpi filtered read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: 46hpi.filtered.tsv - 46hpi filtered read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: 38hpi.normalized.tsv - 38hpi normalized read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: 42hpi.normalized.tsv - 42hpi normalized read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: 46hpi.normalized.tsv - 46hpi normalized read counts in matrix format, where rows are samples and columns are genes
Supplementary_files_format_and_content: all.normalized.tsv - all normalized read counts from all time points in matrix forrmat, where rows are samples and columns are genes

Submission date

Sep 07, 2017

Last update date

May 15, 2019

Contact name

Daniel E. Neafsey

E-mail(s)

[email protected]

Organization name

Broad Institute

Street address

415 Main St.