Unknown,Transcriptomics,Genomics,Proteomics

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Single-cell 5' Gene Expression and V(D)J Amplification (10X Genomics) of splenic CD4+ T cells from Plasmodium chabaudi infected mice

ABSTRACT: To associate gene expression profiles with specific features of the splenic CD4+ T-cell repertoire generated during a blood stage P. chabaudi infection, we obtained both transcriptomic and T-cell receptor data from 2 infected mice at 7 days following infection.

INSTRUMENT(S): NextSeq 550

ORGANISM(S): Mus musculus

SUBMITTER: Catherine Sutherland

PROVIDER: E-MTAB-9691 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Erythrocytes were infected with Plasmodium chabaudi chabaudi AS strain expressing GFP. After 2 or 3 passages, a suspension of 100,000 infected erythrocytes in 1X-PBS (adjusted volume of 200µl) were injected i.p. in wild-type C57BL/6j mice and Homozygous mutant Ighm tm1Cgn mice (lacking mature B-cells). As a control, wild-type C57BL/6j mice and Homozygous mutant Ighm tm1Cgn mice were injected i.p with PBS. There were 3 mice (biological replicates) for each of the 4 experimental conditions. Parasitemia was monitored by flow cytometry and PCR weekly. 117 days post infection, mice were euthanized in CO2 chamber and femur and tibia bones were collected in 1X-HBSS/2% FBS. The cells were removed from the bones and the erythrocytes lysed by hypotonic lysis. Cells (20 million) were antibody stained and the 100K cells (Live/CD11b+/Ly6G-/F4/80+) were sorted in 1X-HBSS/20% FBS. For each sample. 50,000 viable cells were pelleted and lysed with cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and incubated in ice for 3 minutes. The lysaste was washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin. Nuclei were pelleted at 500 RCF for 10 min at 4 degrees celcius. The supernatant was discarded and the pellet was resuspended in 50 uL of transposition mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O). The reaction was incubated for 30 minutes in a thermomixer with 1000 RPM mixing. Zymo DNA Clean and concentrator kit (cat# D4014) was used as a cleanup step. The DNA was amplified at 98 degrees C for 30 seconds, then 13 cycles of: 98 degrees C for 10 seconds, 63 degrees C for 30 seconds, 72 degrees C for 1 minute.The library was evaluated in an Agilent TapeStation system and the DNA concentration evaluated by Qubit. Sequencing was performed on Illumina Nextseq 500 with Buffer cartridge v2: Ref: 15057941, High output reagent cartridge v2: Ref: 15057934, NextSeq accessory Box v2: Ref: 15058251, and High output Flow Cell cartridge v2.5: Ref: 20022408

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Single-cell 5' Gene Expression and V(D)J Amplification (10X Genomics) of splenic CD4+ T cells from Plasmodium chabaudi infected mice

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