Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ATAC-seq of macrophages from wild-type mice and Homozygous mutant Ighm tm1Cgn mice (lacking mature B-cells) following infection with Plasmodium chabaudi chabaudi AS

ABSTRACT: 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

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Daniel Caffrey

PROVIDER: E-MTAB-10086 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Patients received standardized coartem therapy. Briefly, each pill contains 20 mg of artemether and 120 mg of lumefantrine. The dose and total coarse of tablets is based on the patients body weight. Blood was drawn from patients prior to anti-malarial treatment (day 0) and after treatment (day42 for children and day 28 for adults). Peripheral Blood Mononuclear Cells were isolated from all patients. Briefly, for PBMC isolation, whole blood from was diluted 1:2 in sterile PBS and overlaid on Ficoll Paque-PLUS in 50 mL conical tubes and centrifuged 800xg for 15 minutes at 25°C with no brake. Resulting buffy coats were collected and washed twice in RPMI 1640 medium, and then cells were treated with Red Blood Cell Lysis Buffer for 3 minutes at room temperature. Cells were washed in RPMI and counted on a hemacytometer. Monocytes were purified from PBMC using a miltenyi Pan Monocyte Isolation negative selection kit according to the manufacturer's instructions. 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

Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other

Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon macrophages. Infected and control (24 h incubation) macrophage mRNA samples were subjected to two rounds of amplification using the MessageAmp aRNA kit (Ambion) and manufacturer's instructions. Approximately 400 ng of macrophage mRNA was used as template in the first round of amplification, yielding 8.1 ug of infected macrophage aRNA and 7.8 ug of control macrophage aRNA. Approximately 1 ug of first-round macrophage aRNA was used as template in the second round of amplification, yielding 81.2 ug of infected macrophage aRNA and 83.3 ug of control macrophage aRNA. Infected and control macrophage aRNA samples were visualized on agarose gels to check quality and quantity. Except for the amounts of aRNA and reverse transcription (RT) primer used, macrophage target labeling reactions and microarray hybridizations were identical. Macrophage target syntheses used 5 ug of second-round aRNA and 5 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5'T18[Q-N]3'), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 72 Cy3, PMT 63 or 64 Cy5 for macrophage study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other

Project description:The discovery of small open reading frames (smORFs) encoding for polypeptides (SEPs; <100aa) highlights that the coding capacity of the genomes has been underestimated. Most ORF-finding algorithms have historically set a minimum threshold length of 100 aa. As consequence, some transcripts encoding for SEPs, were erroneously discarded or classified as non-coding RNAs (ncRNAs). With this experiments we try to experimentally assess the existence and complexity of these small proteins. The experimental design includes: After growing each Mycoplasma for 6h at 37°C, cells were washed twice with PBS and lysed with 700 µl of Qiazol buffer. Then, samples were lysed with 700 µl of Qiazol buffer. RNA extractions were performed by using the miRNeasy mini Kit (Qiagen) following the instructions of the manufacturer. Libraries for RNA-seq were prepared following directional RNA-seq library preparation and sequencing. Briefly, 1 µg of total RNA was fragmented to ~100-150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5’ and 3’ ends of the RNA available for adapter ligation. Samples were further processed using the TruSeq small RNA Sample Prep Kit (ref. RS-200-0012, Illumina) according to the manufacturer's protocol. In summary, 3’ adapters and subsequently 5’ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3’ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit. Finally, size selection of the libraries was performed using 6% Novex® TBE Gels (ref. EC6265BOX, Life Technologies). Fragments with insert sizes of 100 to 130 bp were cut from the gel, and cDNA was precipitated and eluted in 10 µl of elution buffer. Double-stranded templates were cluster amplified and sequenced on an Illumina HiSeq 2000.

Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.

Dataset Information

ATAC-seq of macrophages from wild-type mice and Homozygous mutant Ighm tm1Cgn mice (lacking mature B-cells) following infection with Plasmodium chabaudi chabaudi AS

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure