Oligonucleotides (Tn5ME-A, Tn5ME-B, Tn5MErev) were resuspended in oligo annealing buffer (10 mM Tris−HCl pH 8.0 (Invitrogen, 15568-025), 50 mM NaCl (Invitrogen, AM9759), 1 mM EDTA (Invitrogen, AM9260G)) to a final concentration of 100μM each. Equimolar amounts of Tn5MErev/Tn5ME-A and Tn5MErev/Tn5ME-B were mixed in separate 200 μl PCR tubes. Then, the adaptors were annealed on the PCR machine with the following PCR program (95°C for 5 min first, then the temperature was slowly ramped down to 25°C with the rate of −0.1°C/s, 25°C for 5 min). The Tn5 transposase was assembled with the following components: 0.04 vol. Tn5MErev/Tn5ME-A, 0.04 vol Tn5MErev/Tn5ME-B, 0.4 vol. 100% glycerol (Sigma-Aldrich, G9012), 0.3048 vol 2× dialysis buffer (100 mM HEPES−KOH (HEPES: Sigma-Aldrich, H3375; KOH: Sigma-Aldrich, 484016) at pH 7.2, 0.2 M NaCl (Invitrogen, AM9759), 0.2 mM EDTA (Invitrogen, AM9260G), 2 mM DTT (Thermo Fisher Scientific, 20291), 0.2% Triton X-100 (Sigma-Aldrich, T8787), 20% glycerol (Sigma-Aldrich, G9012)), 0.043 vol. pure Tn5 (46.55 μM), 0.1722 vol. water (Invitrogen, AM9932). The reagents were mixed thoroughly but gently, and the mixture was left on the bench at RT for 1 h to allow annealing of oligos to Tn5.
Am9260g
Manufactured by Thermo Fisher Scientific
Sourced in Australia
The AM9260G is a laboratory instrument designed for molecular biology applications. It is capable of amplifying DNA or RNA sequences using the polymerase chain reaction (PCR) technique. The device provides precise temperature control and cycling capabilities to facilitate the PCR process. Further details on the specific features and intended applications of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Am9759, by Thermo Fisher Scientific (8 mentions)
H3375, by Merck Group (6 mentions)
Am9932, by Thermo Fisher Scientific (6 mentions)
G9012, by Merck Group (6 mentions)
T8787, by Merck Group (6 mentions)
Matrigel, by Corning (3 mentions)
Am9760g, by Thermo Fisher Scientific (3 mentions)
Y 27632, by Selleck Chemicals (3 mentions)
Vitronectin xf coated, by STEMCELL (3 mentions)
Ab0626, by Thermo Fisher Scientific (3 mentions)
Accutase, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Cls431750 50ea, by BD (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Vitronectin, by Thermo Fisher Scientific (1 mentions)
Essential 8 medium, by Thermo Fisher Scientific (1 mentions)
Macsquant x, by Miltenyi Biotec (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
27 protocols using am9260g
1
Tn5 Transposome Assembly Protocol
2
Tn5 Transposome Assembly Protocol
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Oligonucleotides (Tn5ME-A, Tn5ME-B, Tn5MErev) were resuspended in oligo annealing buffer (10 mM Tris–HCl pH 8.0 (Invitrogen, 15568-025), 50 mM NaCl (Invitrogen, AM9759), 1 mM EDTA (Invitrogen, AM9260G)) to a final concentration of 100 μM each. Equimolar amounts of Tn5MErev/Tn5ME-A and Tn5MErev/Tn5ME-B were mixed in separate 200 μl PCR tubes. Then, the adaptors were annealed on the PCR machine with the following PCR program (95°C for 5 min first, then the temperature was slowly ramped down to 25°C with the rate of −0.1°C/s, 25°C for 5 min). The pA–Tn5 transposase was assembled with the following components: 0.04 vol. Tn5MErev/Tn5ME-A, 0.04 vol Tn5MErev/Tn5ME-B, 0.4 vol. 100% glycerol (Sigma-Aldrich, G9012), 0.3116 vol. 2× dialysis buffer (100 mM HEPES−KOH (HEPES: Sigma-Aldrich, H3375; KOH: Sigma-Aldrich, 484016) at pH 7.2, 0.2 M NaCl (Invitrogen, AM9759), 0.2 mM EDTA (Invitrogen, AM9260G), 2 mM DTT (Thermo Fisher scientific, 20291), 0.2% Triton X-100 (Sigma-Aldrich, T8787), 20% glycerol (Sigma-Aldrich, G9012)), 0.0362 vol pure pA–Tn5 (55.55 μM), 0.1722 vol water (Invitrogen, AM9932). The reagents were mixed thoroughly but gently, and the mixture was left on the bench at RT for 1 h to allow annealing of oligos to pA–Tn5.
3
Tn5 Transposome Preparation Protocol
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Oligonucleotides (T7-Tn5ME, Tn5MErev) were resuspended in oligo annealing buffer (10 mM Tris−HCl pH 8.0 (Invitrogen, 15568-025), 50 mM NaCl (Invitrogen, AM9759), 1 mM EDTA (Invitrogen, AM9260G)) to a final concentration of 100 μM each. Equimolar amounts of Tn5MErev/T7-Tn5ME were mixed in separate 200 μl PCR tubes. Then, the adaptors were annealed on the PCR machine with the following PCR program (95°C for 5 min first, then the temperature was slowly ramped down to 25°C with the rate of −0.1°C/s, finally 25°C for 5 min). The T7−pA−Tn5 transposase was assembled with the following components: 0.08 vol. Tn5MErev/T7-Tn5ME, 0.4 vol. glycerol (Sigma-Aldrich, G9012), 0.3116 vol. 2× dialysis buffer (100 mM HEPES−KOH (HEPES: Sigma-Aldrich, H3375; KOH: Sigma-Aldrich, 484016) at pH 7.2, 0.2 M NaCl (Invitrogen, AM9759), 0.2 mM EDTA (Invitrogen, AM9260G), 2 mM DTT (Thermo Fisher scientific, 20291), 0.2% Triton X-100 (Sigma-Aldrich, T8787), 20% glycerol (Sigma-Aldrich, G9012)), 0.0362 vol. pure pA–Tn5 (55.55 μM), 0.1722 vol. water (Invitrogen, AM9932). The reagents were mixed thoroughly but gently, and the mixture was left on the bench at RT for 1 h to allow annealing of oligos to pA–Tn5.
4
Generation and Maintenance of iPSCs
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Isogenic human iPSC line WTC11 and GRN−/− iPSC line were generated by Dr. Bruce R. Conklin, as previously described (Miyaoka et al., 2014 (link)). GRN−/− iPSC and GRN−/− NGN2 iPSC were engineered (Figure S2 F) and provided by Dr. Michael E. Ward (NIH) as previously described (Wang et al., 2017 (link)). iPSCs were cultured and maintained in Essential 8 Medium (Gibco, A1517001) on 6-well cell culture plates (Olympus, 25-105) coated with Vitronectin (Gibco, A14700) in DPBS. iPSCs were dissociated and passaged using EDTA (Invitrogen, AM9260G) in DPBS. This work was approved by UCSF GESCR Committee.
5
Flow Cytometry Analysis of Immune Cells
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Single-cell suspension was generated (1 × 106 cells in PBS (100 ul)) from MOC22 tumors, spleens or DLNs of mice. Then, cells were first stained with Zombie aqua (Biolegend #423102, 1:500, 15 min at room temperature (RT) in PBS). After washing with 1 mL of PBS, cells were resuspended in FACS buffer (0.5% FBS and 1 mM of EDTA (Invitrogen #AM9260G) in PBS). Treatment with Fc-block (BD Pharmingen #553142, 1:100, 15 min at 4°C) was performed, then appropriate cell surface fluorophore-conjugated anti-mouse antibodies (CD45.2; clone 104, CD11c; clone N418, CD80; clone 16–10A1, CD86; clone GL-1, CD3e; clone 17A2, CD8a; clone 53–6.7, CD4; RM4-5, CD137; 17B5) were added at 1:200, incubated for 20 min at 4°C. Washing with 1 mL of FACS buffer was performed, and cells were analyzed by flow cytometry (Miltenyi MACSQuantX). FlowJo v10.6.2 (BD) software was used to analyze.
6
Characterizing Photosensitive Hydrogels in Matrigel
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A liquid drop of HCC-Gelatin was added onto freshly prepared 100% Matrigel (Corning 354230) solid drops or onto 100% Matrigel drops prepared 2 days before treatment and maintained in DMEM/F12 (ThermoFisher Scientific) at 37 °C. Treated samples were incubated at 37 °C for 15 min. The volume ratio between photosensitive hydrogels and solid Matrigel drops was 2:1. Hydrogels were fabricate within the pre-existing Matrigel with drops by using Scientifica 2-Photon microscope according to our previous study9 (link). For AFM measurements, Matrigel was depolymerized by using cold PBS-EDTA (0.5 mM EDTA, Invitrogen #AM9260G) under gentle agitation overnight at 4 °C as previously shown9 (link). All the samples were analysed by using an Atomic Force Microscope, mounted on an Inverted Optical Microscope (XEBio, Park Systems, Korea) as previously reported9 (link).
7
Quantification of Viral Genome Titers
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Samples were lysed using 10× lysis buffer (Tris-HCl 500 mM [T1503, Sigma-Aldrich], Triton X-100 1% [v/v] [9002-93-1, Sigma-Aldrich], MgCl2 20 mM [SLBP3227V, Sigma-Aldrich] [pH 7.5]) for 1 h at 37°C with gentle agitation. Lysed samples were kept at −80°C for storage. For the determination of vg titer; first, DNase I was added to lysed samples at 0.165 mg/mL (10104159001, Roche) and incubated at 37°C for 16 h in a DNase buffer (5 mM CaCl2, 5 mM MgCl2, 50 mM Tris-HCl [pH 8.0]). EDTA 30 mM (AM9260G, Invitrogen) was then added to each tube at and incubated at 70°C for 30 min to inactivate DNase I. Then, Proteinase K (2 mg/mL, EO0491, Thermo Fisher Scientific) was added and incubated at 55°C for 2 h and inactivated at 95°C, 15 min. qPCR reaction was set up using a set of primers specific to a region within the eGFP gene using an iTaq Universal SYBR Green Supermix (Bio-Rad) and the recommended thermocycling profile in a CFX384 Touch Real-Time PCR Detection System.
8
Generation and Maintenance of Human iPSC Lines
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The use of human iPSCs in the present work was approved by the competent authorities (Commission on Guarantees concerning the Donation and Use of Human Tissues and Cells of the Carlos III Spanish National Institute of Health). A control human iPSC line generated from a healthy individual (codename FiPS Ctrl1-mR5F-6, registered in the National Stem Cell Bank, Carlos III Spanish National Institute of Health) was used for the generation of human iPSC-CCNP knock-outs (KOs). Cells were cultured in 1:100 Matrigel (354277, Corning)-coated dishes and routinely maintained in mTeSR1 medium (Stem Cell Technologies). The medium was changed every day. Cells were split by dissociation with 0.5 mM ethylenediaminetetraacetic acid (EDTA) (AM9260G, Invitrogen) for 2 min at 37 °C and seeded on Matrigel-coated plates. Cell lines were used at passages 20–45.
9
Differentiation of THP-1 Monocytes to Macrophages
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THP-1 monocytes were obtained from ATCC® (Manassas, VA, USA). Cells were maintained in RPMI 1640 medium (ATCC®) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GIBCO®, Waltham, Massachusetts, USA) and 0.05 mM 2-mercaptoethanol (Sigma®, St. Louis Missouri, USA) during 5 days in a humidified incubator at 37°C with 5% of CO2. THP-1 monocytes were differentiated to macrophage-like cells as previously reported [23 (link)]. Briefly, cells were exposed to 50 ng/mL phorbol myristate acetate (PMA) (Sigma®) during 24 h and then incubated for additional 24 h in the absence of PMA before further treatment. Macrophage-like THP1 cells were harvested by washing them with cold PBS (Gibco®) with 1% FBS and then exposed to cold 25 mM EDTA (Invitrogen®, AM9260G) for 10 min in ice. Cells were gently removed, suspended, and washed with RPMI 1640 medium containing 10% FBS. Cells were counted, and the viability was assessed using trypan blue (Sigma®, T8154-100ML). In all experiments, we used microscopy to confirm that PMA treatment induced the expected morphological changes; we also evaluated CD14 and CD11 expression by flow cytometry using anti-CD14 (BD Biosciences, La Jolla, CA, USA) and anti-CD11b (BioLegend®, San Diego, CA, USA) antibodies.
10
Genomic DNA Extraction from FFPE and Frozen Nuclei
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For FFPE-ATAC samples, single nuclei were isolated following the nuclei isolation protocol stated in the section on nuclei isolation from FFPE tissue sections. For frozen samples, nuclei were isolated following the nuclei isolation protocol in the section on standard ATAC-seq on frozen tissue. For genomic DNA purification, 1 million isolated nuclei were spined down at 3000g for 10 min and then resuspended with 100 µL of lysis buffer (50 mM Tris-HCl at pH 7.5 [Invitrogen 15567027], 1 mM EDTA [Invitrogen AM9260G], 1% SDS [Invitrogen 1553-035], 200 mM NaCl [Invitrogen AM9759], and 200 µg/mL Proteinase K [Thermo Fisher Scientific EO0491]). Nuclei suspension was incubated overnight at 65°C with 1200 rpm shaking in a heat block. On the next day, the mixture was purified with a Qiagen MiniElute purification kit (Qiagen 28004) and eluted in 20 µL of elution buffer. Purified genomic DNA was measured and was run on a 1.5% agarose gel (Lonza 50004) to check size distribution.
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