Somatosensory and motor cortex from wild-type animals was dissected and dissociated at P1, P7 and P21. For consistency, we collected the same cortical regions as described above for the bulk profiling of transgenically labeled neurons. Each library was made from tissue pooled from at least eight animals, and a balanced sex ratio was used. Tissue dissociation was performed as described above, and live cells were isolated by FACS sorting as DAPI-negative, Vybrant DyeCycle Ruby (Thermo Fisher Scientific)-positive events. Libraries were prepared using the 10x Genomics Chromium Single Cell 3′ kit v2 according to the manufacturer’s protocol.
Vybrant dyecycle ruby
Manufactured by Thermo Fisher Scientific
The Vybrant DyeCycle Ruby is a fluorescent dye used for nucleic acid staining in flow cytometry applications. It binds to DNA and emits fluorescence when excited by a suitable light source, allowing the detection and analysis of cells based on their DNA content.
Automatically generated - may contain errors
Lab products found in correlation
Ez prep buffer, by Merck Group (4 mentions)
Ma900 cell sorter, by Sony (3 mentions)
Bovine serum albumin (bsa), by New England Biolabs (2 mentions)
Glass dounce homogenizer, by Merck Group (2 mentions)
Influx cell sorter, by BD (2 mentions)
Moflo astrio, by Beckman Coulter (2 mentions)
Infacsmax cell dissociation buffer, by AMS Biotechnology (2 mentions)
Filcon syringe, by BD (2 mentions)
Moflo xdp cell sorter, by Beckman Coulter (2 mentions)
D8938, by Merck Group (1 mentions)
7 aad, by BioLegend (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apc, by BD (1 mentions)
Nylon mesh, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Facsmelody, by BD (1 mentions)
Yo pro 1 dye, by Thermo Fisher Scientific (1 mentions)
Liberase tl, by Merck Group (1 mentions)
Dnase 1, by Sinopharm (1 mentions)
Hepes, by Merck Group (1 mentions)
17 protocols using vybrant dyecycle ruby
1
Profiling Somatosensory and Motor Cortex Cells
2
Nuclear Isolation and Sorting for Genomic DNA Extraction
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Nuclear isolation and sorting were performed as described previously.25 ,68 (link) Dissected cortex tissue was first homogenized using a glass Dounce homogenizer (Sigma-Aldrich; D8938) with 20 strokes of pestle A followed by 20 strokes of pestle B in 2 mL of ice-cold EZ-PREP buffer ((Sigma-Aldrich). Sample was decanted into a new tube with additional 2 mL of cold EZ-PREP buffer on ice and centrifuged (500g, 4°C). The supernatant was decanted, and the nuclei pellet was resuspended in 4 mL of ice-cold Nuclei Suspension Buffer (NSB: 100 mg/mL BSA (New England Biolabs) and 3.33 mM Vybrant DyeCycle Ruby (Thermo Fisher) in PBS). The sample was again centrifuged at 500g for 5 min at 4°C, the supernatant was decanted, and the nuclei were resuspended in 1 mL of NSB. Samples were passed twice through a 35-μM cell strainer before flow sorting using the Sony MA900 Cell Sorter (Sony Biotechnology) at the Broad Institute flow cytometry core. See Figure S7 B for example FACS gating. Nuclei were sorted into DNAdvance lysis buffer, and the genomic DNA was purified according to the manufacturer’s protocol (Beckman Coulter).
3
Isolation and Purification of Nuclei
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Nuclei were isolated from the cortex and the mid-brain as previously described (Levy et al., 2020 (link)). Briefly, dissected cortex and mid-brain were homogenized using a glass Dounce homogenizer (Sigma-Aldrich; D8938) with 20 strokes using pestle A followed by 20 strokes from pestle B in 2 mL of ice-cold EZ-PREP buffer (Sigma-Aldrich; NUC-101). Samples were then decanted into a new tube containing an additional 2 mL of EZ-PREP buffer on ice. After 5 min, homogenized tissues were centrifuged for 5 min at 500 g at 4°C. The nuclei pellet was resuspended in 4 mL of ice-cold Nuclei Suspension Buffer (NSB) consisting of 100 μg/mL BSA (NEB; B9000S) and 3.33 μM Vybrant DyeCycle Ruby (Thermo Fisher; V10309) in PBS followed by centrifugation at 500 g for 5 min at 4°C. After centrifugation, the supernatant was removed, and nuclei were resuspended in 1-2 mL of NSB, passed through 35-μm cell strainer, followed by flow sorting using the Sony MA900 Cell Sorter (Sony Biotechnology) at the Broad Institute flow cytometry core. See Figure S5 A for example FACS gating. Nuclei were sorted into DNAdvance lysis buffer, and the genomic DNA was purified according to the manufacturer’s protocol (Beckman Coulter; A48705).
4
Comprehensive B-cell Immunophenotyping and Apoptosis
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For splenic, LN, and peritoneal B-cell identification and relative quantification, cells were isolated from spleen, LN, or peritoneal lavage and stained in FACS buffer at recommended concentrations for 15 min. For apoptosis assay, cells were stained with Annexin V-APC (BD Biosciences, San Jose CA) according to manufacturer’s staining protocol, and counterstained with 7AAD (Biolegend). For cell cycle analysis, cells were harvested into complete medium with 1:20 7AAD and 1:500 Vybrant DyeCycle Ruby (Thermo Fisher) and incubated for at least 30 min at 37°C in the dark. Cells were analyzed using an LSRII on low or medium speed and by first gating on live, singlet cells. A minimum of 10,000 events was recorded for cellular assays and 500,000 for immunophenotyping.
5
Cell Cycle and Apoptosis Assay
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For cell cycle assay, cell suspensions of M‐204 sh‐Ctrl and sh‐ILF2 (5 × 105 cells in 0.5 ml complete RPMI medium) were filtered through a nylon mesh (40 μm, BD Falcon) to remove cell clumps. The cells were stained with Vybrant® DyeCycle™ Ruby in 5 μM final concentration (Cat# V10309, Thermo Fisher Scientific) at 37˚C for 30 min in the dark. The cells were analysed using BD FACS Melody (BD Biosciences, Franklin Lakes, NJ) based on the fluorescence emission intensity, which was correlated with the DNA content. Apoptosis was measured using Annexin V‐PE Apoptosis Detection Kit I (Cat# 559763, BD Biosciences). A total of 1 × 105 cells of M‐204 sh‐Ctrl or sh‐ILF2 (in 100 μl binding buffer) were stained with 5 μl Annexin V‐PE and 5 μl 7‐AAD for 15 min at RT in dark. Four hundred microliters of binding buffer was then added to the samples and analysed using BD FACS Melody.
6
Apoptosis Screening in Spermatozoa
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Spermatozoa labeled with the YO-PRO-1 dye are undergoing or have undergone apoptosis, as the YO-PRO-1-dye readily enters cells through defects in the membrane (Fig. 1 ). YO-PRO-1-negative spermatozoa are characterized by low DNA fragmentation, as demonstrated by TUNEL [54 (link)]. Initially, the spermatozoa were stained with 0.2 µM of the YO-PRO-1 dye (Thermo Fisher Scientific) and with 1 µg/ml of the Hoechst 33342 dye to exclude debris, then diluted in MHF-10 media for 30 min at room temperature in the dark. For the validation, spermatozoa were also stained with 0.2 µM of the YO-PRO-1 dye and 5 µM of the Vybrant Dye Cycle Ruby (Thermo Fisher Scientific) for 30 min at room temperature in the dark. After staining, spermatozoa were immediately sorted using a BD Influx cell sorter. YO-PRO-1-positive sorted sperm populations were labeled as YOPRO+, whereas YO-PRO-1-negative sorted sperm populations were labeled as YOPRO−.
7
Isolation of Neuronal Nuclei for Genomic Analysis
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At DIV 1, 15 μL of lentivirus was added at 10:10:1 ratio of
N-terminal:C-terminal:nicking sgRNA. At DIV 14, neuronal nuclei were isolated
using the EZ-PREP buffer (Sigma D8938) following the manufacturer’s
protocol. All steps were performed on ice or at 4 °C. Media was removed
from dissociated cultures, and cultures were washed with ice-cold PBS. PBS was
aspirated and replaced with 200 μL EZ-PREP solution. Following a 5-minute
incubation on ice, EZ-PREP was pipetted across the surface of the well to
dislodge remaining cells. The sample was centrifuged at 500 g for 5 minutes, and
the supernatant removed. Samples were washed with 200 μL EZ-PREP and
centrifuged again at 500 g for 5 minutes. Samples were resuspended with gentle
pipetting in 200 μL ice-cold Nuclei Suspension Buffer (NSB) consisting of
100 μg/mL BSA and 3.33 μM Vybrant DyeCycle Ruby (Thermo Fisher) in
1×PBS, then centrifuged at 500 g for 5 minutes. The supernatant was
removed and nuclei were resuspended in 100 μL NSB and sorted into 100
μL Agencourt DNAdvance lysis buffer using a MoFlo Astrios (Beckman
Coulter) at the Broad Institute flow cytometry facility. Genomic DNA was
purified according to the manufacturer’s Agencourt DNAdvance
instructions.
N-terminal:C-terminal:nicking sgRNA. At DIV 14, neuronal nuclei were isolated
using the EZ-PREP buffer (Sigma D8938) following the manufacturer’s
protocol. All steps were performed on ice or at 4 °C. Media was removed
from dissociated cultures, and cultures were washed with ice-cold PBS. PBS was
aspirated and replaced with 200 μL EZ-PREP solution. Following a 5-minute
incubation on ice, EZ-PREP was pipetted across the surface of the well to
dislodge remaining cells. The sample was centrifuged at 500 g for 5 minutes, and
the supernatant removed. Samples were washed with 200 μL EZ-PREP and
centrifuged again at 500 g for 5 minutes. Samples were resuspended with gentle
pipetting in 200 μL ice-cold Nuclei Suspension Buffer (NSB) consisting of
100 μg/mL BSA and 3.33 μM Vybrant DyeCycle Ruby (Thermo Fisher) in
1×PBS, then centrifuged at 500 g for 5 minutes. The supernatant was
removed and nuclei were resuspended in 100 μL NSB and sorted into 100
μL Agencourt DNAdvance lysis buffer using a MoFlo Astrios (Beckman
Coulter) at the Broad Institute flow cytometry facility. Genomic DNA was
purified according to the manufacturer’s Agencourt DNAdvance
instructions.
8
Cardiac Vesicle Isolation from Mice
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CardRED mice hearts were cut into small pieces and digested in DPBS with 0.25 mg ml−1 of Liberase TL (Sigma-Aldrich), 20 μg ml−1 DNase I (Sinopharm Chemical Reagent) and 10 mM HEPES (Sigma-Aldrich) for 15 min at 37 °C. Then, tissue suspensions were acquired by gentle pipetting. We serially centrifuged these suspensions at 50g and 300g and discarded the pellet. The supernatant was then centrifuged at 1,000g and washed with DPBS. Pellets were resuspended with 200 μl DPBS. We used the endogenous expression of tdTomato, CD31 and Vybrant DyeCycle Ruby (Thermo Fisher Scientific, 10 μM) to define cardiac vesicles. The gating strategy for cardiac vesicles is shown in Extended Data Fig. 6c . The material was sorted in a Beckman MoFlo Astrios EQ (Beckman) with a 100-μm nozzle.
9
Isolating Olig1-Expressing Cells from Zebrafish Embryos
10
Nuclei Isolation from Brain Tissue
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Dissected brain tissue was homogenized in 2 ml of ice-cold EZ-PREP buffer (Sigma-Aldrich) using a glass Dounce homogenizer (Sigma-Aldrich) with 20 strokes of pestle A followed by 20 strokes of pestle B. Homogenized samples were then decanted into a new tube containing an additional 2 ml of EZ-PREP buffer on ice. After 5-min incubation, samples were centrifuged for 5 min at 500g at 4 °C. The nuclei pellet was resuspended in 4 ml of ice-cold nuclei suspension buffer (NSB) consisting of 100 µg ml−1 BSA (New England Biolabs) and 3.33 µM Vybrant DyeCycle Ruby (Thermo Fisher Scientific) in cold PBS. Nuclei were then centrifuged at 500g for 5 min at 4 °C. The nuclei were resuspended in 1–2 ml of NSB, passed through a 35-µm cell strainer and then sorted using a MA900 Cell Sorter (Sony Biotechnology) at the Broad Institute Flow Cytometry Core. See Supplementary Fig. 17 for FACS gating strategy. Nuclei were sorted into DNAdvance lysis buffer (Beckman Coulter), and gDNA was purified.
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