Dataset Information

Increasing sustainability in palaeoproteomics by optimizing digestion times for large-scale archaeological bone analyses

ABSTRACT: # Description of data generation and experimental procedure Six bones from La Draga (Spain, Holocene, samples LD_01 to LD_06) and Baiyisha Karst Cave (China, Pleistocene, samples BKC_07 to BKC_12) were sampled for the purpose of this study. Initial sampling was divided into three sub-samples for the three digestion duration tested here (site code_sample number_3h, site code_sample number_6h and site code_sample number_18h). Samples were then processed according to the ZooMS protocol: they were demineralised in 0.6 M hydrochloric acid (HCl) for 24 hours. The HCl supernatant was then removed and samples were rinsed thrice in 100 μL ammonium bicarbonate (50 mM, NH4HCO3, hereafter AmBic) for subsequent gelatinisation in a final volume of 100 μL AmBic for one hour at 65C. Following gelatinisation, the 100 μL AmBic solution was transferred to a new microtube, to which 0.8 μg trypsin (Promega) was added for incubation at 37 °C, with mild agitation at 300 rpm (VWR, Thermal Shake lite). Digestion occurred for either 3, 6, or 18 hours. To stop trypsin digestion, 2 μL of 5% trifluoroacetic acid (TFA) was added to each sample. The digested extracts were then split in two parts for separate analyses via matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-ToF MS) and liquid-chromatography tandem mass spectrometry (LC-MS/MS). To assess any potential contamination by non-endogenous peptides, we performed extraction of laboratory blanks alongside the samples for each enzymatic digestion condition.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Bovinae

TISSUE(S): Bone

SUBMITTER: Frido Welker

LAB HEAD: Frido Welker

PROVIDER: PXD045027 | Pride | 2024-03-14

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20230217_EXPL5_EVO5_GT_100SPD_15k_Blank_004.raw	Raw
	20230217_EXPL5_EVO5_GT_100SPD_15k_Blank_005.raw	Raw
	20230217_EXPL5_EVO5_GT_100SPD_15k_Blank_006.raw	Raw
	20230217_EXPL5_EVO5_GT_100SPD_15k_P0244.raw	Raw
	20230217_EXPL5_EVO5_GT_100SPD_15k_P0245.raw	Raw

Items per page:

1 - 5 of 45

Similar Datasets

Project description:2×10^7 HL60 cells were treated with 10 uM ATRA for 2 and 5 days, and conducted Hi-C to study the chromosomal architecture. Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 μl lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 μl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight. The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB). For regular 3C ligation, after enzymes inactivation, 10 μl 1 U/μl T4 DNA ligase was added to incubate at 16°C overnight. The interaction was detected by qPCR using the specific 3C primers (Table 1) followed by DNA purification. Hi-C was conducted in PE-150. An iterative method for Hi-C mapping with initial length 30bp, if it fails to map uniquely, the next round of mapping for additional 20 bp continued, this procedure lasts until the full read length reaches 150bp. The Hi-C reads were mapped to the human reference genome (assembly GRCh38) using bowtie2 (v2.2.9). The reads mapped to the same restriction fragment, the reads less than 500bp and PCR duplicates were all removed. Hi-C contact maps were normalized using ICE.

Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

Dataset Information

Increasing sustainability in palaeoproteomics by optimizing digestion times for large-scale archaeological bone analyses

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets