Neuronal Nuclei Isolation and FACS Sorting

Nuclei were isolated from frozen tissue as described above. Before FACSing, nuclei were incubated with recombinant Alexa Fluor 488 anti-NeuN antibody [EPR12763]–neuronal marker (catalog no. ab190195, Abcam, RRID:AB_2716282) at a concentration of 1:500 for 30 min on ice as previously described (79 (link)). The nuclei were run through the FACS at 4°C with a low flow rate using a 100-mm nozzle, and 300,000 Alexa Fluor 488–positive nuclei were sorted. The sorted nuclei were pelleted at 1300g for 15 min and resuspended in 1 ml of ice-cold nuclear wash buffer (20 mM Hepes, 150 mM NaCl, 0.5 mM spermidine, 1× cOmplete protease inhibitors, 0.1% bovine serum albumin) and 10 μl per antibody treatment of ConA-coated magnetic beads (Epicypher) added with gentle vortexing (pipette tips for transferring nuclei were precoated with 1% bovine serum albumin). Protocol can be found at DOI: dx.doi.org/10.17504/protocols.io.4r3l27pejg1y/v1.

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Garza R., Atacho D.A., Adami A., Gerdes P., Vinod M., Hsieh P., Karlsson O., Horvath V., Johansson P.A., Pandiloski N., Matas-Fuentes J., Quaegebeur A., Kouli A., Sharma Y., Jönsson M.E., Monni E., Englund E., Eichler E.E., Gale Hammell M., Barker R.A., Kokaia Z., Douse C.H, & Jakobsson J. (2023). LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification. Science Advances, 9(44), eadh9543.

Publication 2023

ab190195

Alexa fluor 488 Anti antibody Antibody Bovine serum albumin Buffer Cold Cona Frozen Hepes Nacl Neuronal Nuclei Protease inhibitors Spermidine Tissue

Corresponding Organization : Lund University

Other organizations : University of Washington, University of Minnesota Medical Center, Cambridge University Hospitals NHS Foundation Trust, University of Cambridge, Wellcome/MRC Cambridge Stem Cell Institute, Howard Hughes Medical Institute, Institute for Systems Biology

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Variable analysis

independent variables

Incubation of nuclei with recombinant Alexa Fluor 488 anti-NeuN antibody [EPR12763]–neuronal marker (catalog no. ab190195, Abcam, RRID:AB_2716282) at a concentration of 1:500 for 30 min on ice

dependent variables

Number of Alexa Fluor 488–positive nuclei sorted

control variables

Incubation temperature (4°C)
Flow rate (low)
Nozzle size (100-mm)
Nuclear wash buffer composition (20 mM Hepes, 150 mM NaCl, 0.5 mM spermidine, 1× cOmplete protease inhibitors, 0.1% bovine serum albumin)
Precoating of pipette tips with 1% bovine serum albumin
Use of ConA-coated magnetic beads (Epicypher)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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