Tn5 reaction buffer was prepared: 50 mM TAPS (Sigma #T9659-100G), 25 mM MgC12, (Ambion#AM9530G); the pH was adjusted to 8.5 and the solution was sterile filtered. Tn5 dilution buffer was prepared: 50 mM Tris-HC1 pH 7.5 (Sigma #T2944-100ML), 100 mM NaCl (Sigma #S5150-1L), 0.1 mM EDTA (Invitrogen #AM9260G), 50% glycerol (Sigma #G5516-100ML), 0.1% Triton-X100 (Sigma #X100-100ML), and supplemented with fresh 1 mM DTT (Sigma #646563-10x.5ML) before use. To achieve maximum library complexity, the entire sample was processed in several tagmentation reactions with 1 ng input each. cDNA was diluted to 0.2 ng/μl with nuclease-free water and 5 μl (1 ng) per reaction was distributed into a 96-well plate on ice. A mix of 11.25 μl nuclease-free water, 5 μl of 5x Tn5 reaction buffer, 2.5 μl of dimethylformamide (Sigma #D4551-250ML), and 1.25 μl of freshly diluted i7-only transposome (prepared as described below and diluted 1:4.5 in Tn5 dilution buffer) was added. Reactions were incubated for 10 min at 55 °C, then cooled for 1 min on ice. The enzyme was inactivated by addition of 2.5 μl of 1% SDS (Sigma #71736-100ML) for 5 min at room temperature. Next, the volume was brought to 50 μl and the fragmented cDNA was purified with a 1.0x SPRI cleanup, eluting in 17 μl of EB buffer (Qiagen #19086).
Am9260g
Manufactured by Merck Group
The AM9260G is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of the AM9260G is to provide a precise and reliable measurement or analysis capability, but no further details about its intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Am9530g, by Thermo Fisher Scientific (3 mentions)
Ultrapure dnase rnase free water, by Thermo Fisher Scientific (2 mentions)
Suc507, by Avantor (2 mentions)
T8787 50ml, by Thermo Fisher Scientific (2 mentions)
Eb buffer, by Qiagen (1 mentions)
D4551 250ml, by Merck Group (1 mentions)
X100 100ml, by Merck Group (1 mentions)
Protector rnase inhibitor, by Merck Group (1 mentions)
Hiseq x ten pe150 platform, by Illumina (1 mentions)
Fc 121 1030, by Illumina (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Am9760g, by Thermo Fisher Scientific (1 mentions)
Digitonin, by Merck Group (1 mentions)
Am9759, by Thermo Fisher Scientific (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Qubit tube, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Proteinase k, by GeneAll (1 mentions)
6 protocols using am9260g
1
Tn5 Tagmentation of cDNA Libraries
2
Single-Nucleus RNA Sequencing of Frozen Tissue
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A pilot single-nucleus RNA sequencing experiment was undertaken to compare single-cell versus single-nuclear results from a matched sample. The biopsy from a 58-year-old male was collected fresh and divided into 8 segments, evenly distributed to be processed fresh for single cell RNA sequencing as above, and the remainder was flash frozen in liquid nitrogen. The sample was later retrieved from liquid nitrogen and processed on dry ice according to the protocol in88 (link) with a lysis buffer containing: 0.32 mM sucrose (BioShop SUC507.1), 5 mM CaCl2 (VWR, 97062-820), 3 mM MgCl2 (Thermo Fisher AM9530G), 20 mM Tris-HCl pH 7.5 (Thermo Fisher, 15567027), 0.1% TritonX-100 (Sigma Aldrich T8787-50ML), 0.1 mM EDTA pH 8.0 (Thermo Fisher AM9260G), 40 U/ml Protector RNAse inhibitor (Sigma Aldrich 3335399001) in UltraPure DNAse/RNAse-free water (Thermo Fisher 10977015). The nuclei were captured and sequenced using 10X Genomics Single Cell 3’ v3 Reagents as above.
3
Multiplex Tissue Nuclei Profiling
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Approximately 5 mg of tissue from each of the skeletal muscle, spleen, heart, liver, and fat samples was crushed into a fine powder in liquid nitrogen (Supplementary Table 1 ). Pulverized tissues were suspended in 1 mL ice-cold PBS and then gently spun down. The cell pellet was resuspended in 1 mL lysis buffer (50 mM HEPES with pH 7.5 [Life technologies, 15630-080], 140 mM NaCl [Ambion, AM9760G], 1 mM EDTA with pH 8.0 [Ambion, AM9260G], 10% glycerol [Sigma-Aldrich, G7757], 0.5% NP-40 [Roche, 11754599001], and 0.25% Triton X-100 [Amresco, 0694]), followed by isolation of 50,000 nuclei using a previously published protocol with minor modifications79 . Then, the transposition reaction mix (12.5 μL TD buffer, 10 μL ddH2O, and 2.5 μL TDE [Illumina, FC-121-1030]) was added to the isolated nuclei. The reaction system was incubated at 37 °C for 1 h followed immediately by purification with a QIAGEN MinElute PCR Purification Kit (QIAGEN, 28006). The transposed DNA fragments were amplified with the appropriate number of cycles as determined by qPCR. Size selection of the amplified PCR products (100–600 bp fragments) was performed by gel-purification informing previously studies79 –81 (link). Products from the size selection step were quantified and sequenced using an Illumina HiSeq X Ten PE150 platform.
4
Nuclei Extraction and H3K27ac FACT-seq Library Prep
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Aliquots of nuclei with epitope retrieval from human GBM or human CRC samples (100 000 cells/0.5 ml Qubit tube (Invitrogen, Q32856)) were washed once with FACT-seq Antibody buffer (20 mM HEPES(K+) pH 7.6 (HEPES: Sigma-Aldrich, H3375; KOH: Sigma-Aldrich, 484016), 150 mM NaCl (Invitrogen, AM9759), 2 mM EDTA (Invitrogen, AM9260G), 0.5 mM spermidine (Sigma-Aldrich, S2626), 0.05% digitonin (Millipore, 300410), 0.01% IGEPAL® CA-630 (Sigma-Aldrich, 13021-50), 1× protease inhibitors (Sigma-Aldrich, 11873580001), 1% BSA (Miltenyi Biotech MACS, 130-091-376)). H3K27ac FACT-seq sequencing libraries were prepared with same methods as in the mouse samples.
5
Stabilization of Saliva Samples for LAMP
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Stabilization solution was prepared with TCEP-HCl (Sigma-Aldrich, 75259), EDTA (ThermoFisher Scientific, AM9260G), NaOH (Sigma-Aldrich, 72068), Proteinase K (Geneall, 106–101) and Nuclease free water. The final concentration of 2X stabilization solution was 5 mM for TCEP-HCl, 2 mM for EDTA, 29 mM for NaOH and 100 μg/mL for Proteinase K. 6 μL of stabilization solution was added to 1 μL Saliva (Lee Biosolution, 991-05-P, St. Louis, MO, USA) mixed with 5 μL synthetic RNA in nuclease-free water. It was heated at 95 °C for 10-min and 10 μL of it was put into LAMP reaction.
6
Single-Nucleus RNA Sequencing Pilot
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A pilot single-nucleus RNA sequencing experiment was undertaken to compare single cell versus single nuclear results from a matched sample. The biopsy was collected fresh and divided into 8 segments, evenly distributed to be processed fresh for single cell RNA sequencing as above, and the remainder was flash frozen in liquid nitrogen. The sample was later retrieved from liquid nitrogen and processed on dry ice according to the protocol in 74 with a lysis buffer containing: 0.32 mM sucrose (BioShop SUC507.1), 5 mM CaCl2 (VWR, 97062-820), 3 mM MgCl2 (Thermo Fisher AM9530G), 20 mM Tris-HCl pH 7.5 (Thermo Fisher, 15567027), 0.1% TritonX-100 (Sigma Aldrich T8787-50ML), 0.1 mM EDTA pH 8.0 (Thermo Fisher AM9260G), 40 U/ml Protector RNAse inhibitor (Sigma Aldrich 3335399001) in UltraPure DNAse/RNAse-free water (Thermo Fisher 10977015). The nuclei were captured and sequenced using 10X Genomics Single Cell 3' v3
Reagents as above.
Reagents as above.
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