B cells from draining lymph nodes were negatively enriched or not with CD43 (Ly-48) MicroBeads (130-049-801; Miltenyi Biotec), stained, single-cell sorted with a FACSAria II (BD Biosciences) in 96-well plates containing 5 µl of a lysis buffer (TCL Buffer, 1031576; QIAGEN) containing 1% 2-β-mercaptoethanol (M3148; Sigma) and immediately frozen at −80°C. RNA was purified from single cells using magnetic beads (RNAClean XP, A63987; Beckman Coulter) following the manufacturer’s instructions. RNA was eluted from the magnetic beads with 11 µl of a solution containing random primers (14.5 ng/µl, 48190-011; Invitrogen), tergitol (0.5% NP-40 70% in H2O, NP-40S; Sigma-Aldrich), and RNase inhibitor (0.6 U/µl, N2615; Promega) in nuclease-free water (QIAGEN) and incubated at 65°C for 3 min. cDNA was subsequently synthesized by reverse transcription with 7 µl of a solution containing SuperScript III Reverse Transcription, 5× buffer, dithiothreitol (10,000 U, 18080-044; Invitrogen), dNTP (25 µM), and RNase inhibitor (0.6 U/µl, N2615; Promega) in nuclease-free water (QIAGEN) incubated at 1× (42°C for 10 min, 25°C for 10 min, 50°C for 60 min, 94°C for 5 min). cDNA was stored at −20°C or immediately used for antibody gene amplification by nested PCR after addition of 10 µl nuclease-free water.
N2615
Manufactured by Promega
Sourced in United States
The N2615 is a laboratory instrument designed for the extraction and purification of nucleic acids, including DNA and RNA. It utilizes a magnetic bead-based separation technology to efficiently isolate target molecules from complex biological samples. The core function of this product is to provide a reliable and automated method for nucleic acid extraction and purification, which is essential for various downstream applications in molecular biology, genetics, and genomics research.
Automatically generated - may contain errors
Lab products found in correlation
Nuclei ez lysis buffer, by Merck Group (2 mentions)
Am2696, by Thermo Fisher Scientific (2 mentions)
Sybr qpcr premix ex taq 2 tli rnase h, by Takara Bio (2 mentions)
M mlv rt, by Promega (2 mentions)
Facsaria 2, by BD (1 mentions)
Tcl buffer, by Qiagen (1 mentions)
M3148, by Merck Group (1 mentions)
Rnaclean xp, by Beckman Coulter (1 mentions)
Nuclease free water, by Qiagen (1 mentions)
Sytox green dead cell stain, by Thermo Fisher Scientific (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Protein a agarose beads, by Merck Group (1 mentions)
A32965, by Thermo Fisher Scientific (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Myelin removal beads 2, by Miltenyi Biotec (1 mentions)
Np 40, by Thermo Fisher Scientific (1 mentions)
Thermopol buffer, by New England Biolabs (1 mentions)
11 protocols using n2615
1
Single-cell RNA-seq of B cells
2
Isolation and Purification of Adult Mouse Dopaminergic Neurons
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Isolation of adult mouse neurons from fresh ventral midbrain was performed according to a protocol published in Nature Protocols [54 (link)]. During the procedures, the working solutions were supplemented with trehalose (5%, T0167, Sigma) to enhance cell viability [55 (link)]. The digestion medium was further added with DNase I (100 units/mL, LK003172, Worthington). The suspended cells from “release of cells from tissue” step were added with an RNase inhibitor (1:100, N2615, Promega), SYTOX Green Dead Cell Stain (1:1000, S34860, Invitrogen), and NucBlue Live ReadyProbes Reagent (2 drops/mL, R37605, Molecular Probes) to facilitate cell sorting of tdTomato+ live DA neurons on a FACSAria II flow cytometer (BD Biosciences).
3
Nuclei Isolation with Protease Inhibitor
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Nuclei were isolated using Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche) and ribonuclease inhibitor (N2615, Promega and AM2696, Life Technologies).
4
Reverse Transcription and qPCR Analysis
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One microgram of total mRNAs (from tissues or cell culture) was reverse transcribed for 1 h at 37 °C with 5 pmol of random hexamers primers, 200 units reverse transcriptase (M-MLV RT, M1701, Promega), 2 mM dNTPs and 20 units RNAsin (N2615, Promega). One microlitre of a one-tenth dilution of cDNA was used in each qPCR. Except for VegfA and Connexinα43 that were amplified with Taqman chemistry as described in ref. 41 (link), all other reactions were conducted with SYBR qPCR Premix Ex Taq II Tli RNase H+ (TAKRR820W, Takara). Primer pairs are listed in Supplementary Data 3 . For each experiment and primer pairs, efficiency of PCR reactions was evaluated by amplification of serial dilutions of a mix of cDNAs. Relative gene expression was obtained by the ΔΔCt method after normalization to 36b4 (mouse) or PPIB (human).
5
Nuclei Isolation with Protease Inhibitor
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Nuclei were isolated using Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche) and ribonuclease inhibitor (N2615, Promega and AM2696, Life Technologies).
6
Prostate mRNA Quantification by qPCR
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A total of 300 nanograms of total mRNAs from prostate tissues were reverse transcribed for 1 hour at 37°C with 5 pmoles of random hexamer primers, 200 units reverse transcriptase (MMLV RT, M1701, Promega, Madison, Wisconsin), 2 mM dNTPs, and 20 units RNAsin (N2615, Promega). Two microliters of a one-tenth dilution of cDNA was used in each quantitative PCR. PCR reactions were conducted with SYBR qPCR Premix Ex Taq II Tli RNase H+ (TAKRR820W, Takara, Saint-Germain-en-Laye, France). Primer pairs are listed in SI Appendix S1 Table . For each experiment and primer pairs, efficiency of PCR reactions was evaluated by amplification of serial dilutions of a mix of cDNAs. Relative gene expression was obtained by the ΔΔCt method with normalization to expression of 36b4 housekeeping gene.
7
Methanol-Based HIV Flow Cytometry
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The methanol-based HIV-Flow procedure was performed as previously described by Cole et al.40 (link). The detailed protocol can be found here: https://protocols.io/view/methanol-based-hiv-flow-bpedmja6 . In brief, following stimulation, a maximum of 5 × 106 cells per condition were resuspended in PBS and stained with fixable viability stain 510 for 20 min at RT. Cells were then stained with antibodies against cell surface molecules (CD3, CD4, CD8, CD45RO, CD27, PD1) in PBS + 2% FBS for 20 min at 4 °C. After a 5 min-centrifugation step at 4 °C to pre-chill the cells, CD4 cells were vortexed to avoid clumping and 900 μL of ice-cold methanol (−20 °C) was gently added. Cells were fixed/permeabilized in methanol for 15 min on ice. Intracellular p24 staining was performed using a combination of 2 antibodies (p24 KC57-FITC, p24 28B7-APC) (45 min, RT) in PBS/1% BSA (ThermoFisher, #AM2616)/RNAse inhibitor 0.4 U/μL (Promega, #N2615)/DTT 1 mM. All washing steps following methanol permeabilization were done in PBS/0.04% BSA. In all experiments, CD4 T cells from an HIV-negative control were included to set the threshold of positivity.
8
Immunoprecipitation of MIWI and MILI from Mouse Testes
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Mouse testes were collected and homogenized using lysis buffer (20 mM HEPES pH 7.3, 150 mM NaCl, 2.5 mM MgCl2, 0.2 % NP-40, and 1 mM DTT) with protease inhibitor (A32965, Thermo Fisher Scientific) and RNase inhibitor (N2615, Promega). The lysates were pre-cleared using Protein A agarose beads (11134515001, Sigma-Aldrich) at 4 °C for 2 h. Anti-MIWI (2079, Cell Signaling Technology) or anti-MILI (PM044, MBL) antibody together with Protein A agarose beads were added to the lysates and incubated at 4 °C for 4 h. The beads were washed in lysis buffer 5 times. Immunoprecipitated RNAs were isolated from the beads using Trizol reagent (15596026, Thermo Fisher Scientific) for piRNA labeling or small RNA library construction. For protein detection, immunoprecipitated beads were boiled in protein loading buffer for 5 min. Western blotting of MIWI or MILI was performed as described above.
9
Isolating Microglia for Bulk RNA-seq
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To isolate cells for bulk RNA-seq, mice were euthanized with pentobarbital and were subsequently transcardially perfused using ice-cold PBS 1x and the brain and spinal cord were quickly dissected after removal of the meninges and placed in PBS on ice. Microglia/myeloid cell extraction was carried out following a published protocol82 (link). The whole procedure was done on ice with cold buffers containing RNase inhibitors (N2615, Promega). Briefly, the tissue was dounce homogenised in ice-cold PBS with loose and tight pestles. The cell suspension was then transferred through a pre-wet 70-μm cell strainer (BD Falcon). Myelin removal was performed by adding 100 μl Myelin Removal Beads II (Miltenyi Biotec) per dorsal spinal cord and loaded in pre-wet LS columns (Miltenyi Biotec) on a MACS magnet stand. Cells in the flow-through were collected and washed for standard FACS staining.
10
Cytoplasmic and Nuclear RNA Extraction
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After the cells were prepared, the medium was absorbed and discarded, washed twice with PBS, and the cells were scraped off with 400 μL precooled PBS. Then, transferred into 1.5 ml tube, centrifuged at 10,000 rpm at 4 °C for 10 s, and the supernatant was discarded. Resuspend the cells with 400 μL PBS containing RNA enzyme inhibitor (N2615, Promega, USA) (RNA enzyme inhibitors: PBS = 1:20 v/v ratio) and 0.1% NP-40 (85124, Thermo Fisher Scientific, USA) (NP-40: PBS = 1:1000 v/v ratio), put it in ice for 5 min, then vortexed for 5 s. After centrifugation at 10,000 rpm at 4 °C for 20 s, the supernatant was extracted from cytoplasm and transferred into a new tube. At this time, the supernatant was cytoplasm extract, and the precipitate was nucleus. The nuclei were resuspended by 200 μL PBS containing 0.5% NP-40 and RNA enzyme inhibitor (RNA enzyme inhibitors: PBS = 1:20 v/v ratio), and were blown for 10 times, placed on ice for 10 min, and centrifuged for 20 s at 13,000 rpm at 4 °C. The supernatant was the nuclear extract, and nuclear extract and cytoplasmic extract were used for subsequent RT-qPCR experiments according to the above steps.
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