ATACseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE132258 | GEO | 2019/06/06
REPOSITORIES: GEO
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Project description:The goal of this study is to analyse the open chromatin regions via ATACseq in zebrafish embryonic endothelial cells at 26-27hpf using Tg(Kdrl:GFP) transgenic zebrafish embryos. 40000-50000 cells were FAC-sorted directly into 100 µl of 1x HBSS/10 mM HEPES/0.25% BSA buffer for Tagmentation (as described by Buenrostro et al. (2013), Nat. Methods 10, 1213-1218), but with 1.5 µl Tn5 transposase (Illumina) in a 50 µl reaction volume. DNA was purified using the QIAquick PCR purification kit (Qiagen). Fragments were amplified for 16 cycles.
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Project description:3x107 cells were harvested and washed in 30 ml cold 1x TDB. The pellet was resuspended in 300 µl permeabilization buffer with protease inhibitors, 3 µl of 4 mM digitonin was added and incubated for 5 minutes at RT. The cells were pelleted, resuspended in 600 µl isotonic buffer with protease inhibitors and split in two samples, containing 1x107 and 2x107 cells, respectively. The transposition reaction was performed by adding 50 µl of transposition mix to the pellet (25 µl TD (2x reaction buffer from Nextera kit), 25 µl TDE1 (Tn5 transposase from Nextera kit), 22.5 µl nuclease-free water) and incubation for 30 minutes at 37 °C. For the gDNA control, 200 ng of gDNA was treated in the same manner. The DNA samples were purified using Qiagen MinElute PCR Purification Kit and eluted in 10 µl EB (10 mM Tris-HCl, pH 8). The transposed DNA fragments were amplified using the NEBNext® High-Fidelity 2X PCR Master Mix (M0541) supplied with 2.5 µl of 25 µM barcoded primers and amplification for 13 cycles. The libraries were purified using AMPure XP beads (Beckman Coulter) following the manufacturer’s instructions. The library fragment sizes between 150 and 1000 bp were purified from a 6% polyacrylamide gel. Paired-end 76 bp sequencing was carried out using the Illumina NextSeq system with a mid-output NextSeq 500/550 kit according to the manufacturer’s instructions.
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Project description:Trypanosomes were sorted (1 cell, 10 cells, 50 cells) using a FACSaria III (BD Biosciences; precision: single-cell; nozzle: 100 µm). Forward-scatter area (FCS-A) versus side-scatter area (SSC-A) was used to gate the cells. Trypanosomes were sorted in 48-wells plate (Brand) filled with 2.6 µL of lysis buffer (0.01 µL of RNAse inhibitor (Takara) and 1x Lysis buffer (Takara) in RNAse-free water). Immediately after sorting cells were placed on ice for 5 minutes and stored at -80 °C. 50 and single trypanosomes were prepared using SMART-Seq v4 Ultra Low Input RNA Kit (Takara) using one fourth of reagents volumes compared to the supplier instructions. PCR amplification was performed using 26 cycles using supplier recommendations. cDNA was purified using XP beads (Beckman Coulter) and recovered in 15 µL of elution buffer (Takara). Libraries were quantified using the Qubit Hs Assay (Life Technologies) and the qualities of the libraries were further monitored using a Bioanalyzer (Agilent). Similar to what has been published previously 19, 1 ng of cDNA was subjected to a tagmentation-based protocol (Nextera XT, Illumina) using one-quarter of the recommended volumes, 10 minuntes for tagmentation at 55 °C and 1 minute extension time during PCR amplification. Libraries were pooled (96 libraries for NextSeq) and sequencing was performed in paired-end mode for 2 × 75 cycles using Illumina's NextSeq 500.
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Project description:Trypanosomes were sorted (0 cells, 1 cell, 50 cells) using a FACSaria III (BD Biosciences; precision: single-cell; nozzle: 100 µm). Forward-scatter area (FCS-A) versus side-scatter area (SSC-A) was used to gate the cells. Trypanosomes were sorted in 48-wells plate (Brand) filled with 2.6 µL of lysis buffer (0.01 µL of RNAse inhibitor (Takara) and 1x Lysis buffer (Takara) in RNAse-free water). Immediately after sorting cells were placed on ice for 5 minutes and stored at -80 °C. 50 and single trypanosomes were prepared using SMART-Seq v4 Ultra Low Input RNA Kit (Takara) using one fourth of reagents volumes compared to the supplier instructions. PCR amplification was performed using 26 cycles using supplier recommendations. cDNA was purified using XP beads (Beckman Coulter) and recovered in 15 µL of elution buffer (Takara). Libraries were quantified using the Qubit Hs Assay (Life Technologies) and the qualities of the libraries were further monitored using a Bioanalyzer (Agilent). Similar to what has been published previously 19, 1 ng of cDNA was subjected to a tagmentation-based protocol (Nextera XT, Illumina) using one-quarter of the recommended volumes, 10 minuntes for tagmentation at 55 °C and 1 minute extension time during PCR amplification. Libraries were pooled (96 libraries for NextSeq) and sequencing was performed in paired-end mode for 2 × 75 cycles using Illumina's NextSeq 500.
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