Gata6-inducible mES cells were seeded at 1 × 104 cells per square centimeter and treated with 1 µg/mL doxycycline for 36 h prior to harvesting. Immunoprecipitation was performed on 1 × 107 to 2 × 107 cells as described (Vokes et al. 2007 (link)) for three biological replicates versus input samples. Sonication was performed using a Misonix 4000 (28 cycles of 15 sec on and 45 sec off at an intensity of 70%) with a microtip probe (Misonix). The antibodies used are listed in Supplemental Table S7. Libraries were prepared using the TruSeq ChIP sample preparation kit, and the resulting samples were sequenced using the Illumina Genome Analyzer II (Illumina). Data will be deposited into Gene Expression Omnibus and released immediately after publication (GSE69323). Computational analysis details are included in the Supplemental Material.
Microtip probe
Manufactured by Bioventus
Sourced in United States
The Microtip probe is a lab equipment product designed for use in various scientific applications. It serves as a tool for performing precise measurements and analysis within small sample volumes. The core function of the Microtip probe is to provide accurate and reliable data collection in a compact and versatile format.
Automatically generated - may contain errors
3 protocols using microtip probe
1
ChIP-seq analysis of GATA6-induced mES cells
2
Fabrication of Controlled Mimetic Microparticles
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Example 2
Fabrication of Control MPs and CMMPs:
CSC factor-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (Control MP1) were fabricated by a water/oil/water (w/o/w) emulsion technique, Briefly, human CSC conditioned media as the internal aqueous phase with polyvinyl alcohol (PVA) (0.1% w/v) was mixed in methylene chloride (DCM) containing PLGA as the oil phase. The mixture was then sonicated on ice for 30 s using a sonicator with a Microtip probe (Misonix, XL2020, Farmingdale, N.Y., USA). After that, the primary emulsion was immediately introduced into water with PVA (0.7% w/v) to produce a w/o/w emulsion. The secondary emulsion was emulsified for 5 min on a high-speed homogenizer. The w/o/w emulsion was continuously stirred overnight at room temperature to promote solvent evaporation. The solidified microparticles, namely Control MP1, were then centrifuged, washed three times with water, lyophilized and stored at −80° C. To prepare CMMPs, DiO (Invitrogen)-labeled CSCs went through three freeze/thaw cycles. After which, the disrupted CSCs were sonicated for approximately 5 minutes at room temperature along with the Control MP1. After that, the particles were washed three times in PBS by centrifugation. Control MP2 was fabricated by cloaking empty PLGA particles with CSC membranes. Successful membrane coating was confirmed using fluorescent microscopy.
3
Nucleolar Isolation from HeLa Cells
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Nucleoli were isolated after a protocol adapted from Mitrea et al. (2016) (link). 4 × 107 HeLa cells were treated overnight as indicated in the figure legends and collected by trypsinization. Cells were washed in 10 ml PBS and centrifuged at 400 g for 5 min. Cell pellets were resuspended in five pellet volumes of nucleolar isolation buffer (NIB; 10 mM Tris, 2 mM MgCl2, and 0.5 mM EDTA) with complete protease inhibitor (Roche) and incubated at RT for 2 min and then on ice for 10 min. Plasma membranes were disrupted by the addition of 10% (vol/vol) IGEPAL to a final concentration of 1% and mixed by gentle pipetting. Nuclei were isolated by centrifugation at 500 g for 3 min. The supernatant was removed and stored as the cytoplasmic fraction, and the pellets were washed in 10–15 pellet volumes of NIB and 1% IGEPAL. Nuclear pellets were then resuspended in 10 pellet volumes of NIB and sonicated on ice at 20% power for 12 cycles of 1 s on followed by 5 s off on an XL 2020 sonicator fitted with a microtip probe (Misonix). The samples were centrifuged, and the supernatant was stored as the nucleoplasmic fraction. The pellet was washed once more with 10–15 pellet volumes of NIB and finally resuspended in 50–100 µl NIB.
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