All steps and buffers on ice
1. Homogenize tissue in HB (for the developing mammalian brain - by trituration (resuspending with a pipette), use douncing if necessary, cooled down micropestles can be used to dounce in an epi) and let stand on ice 5 min.
modification when cytoplasm is collected for ribo-seq in parallel: no Superasin during the first wash (it inhibits RNAse activity)
2. Removal of unlysed tissue by low speed centrifugation 100g 1 min (alternatively strain, but many nuclei are lost by straining; adjust centrifugation conditions if needed).
3. Pellet the nuclei 400g 4 min (this can be optimized, alternatively 500g 5 min).
4. Wash the nuclei with HB (we currently wash 1-2 times, the first wash is the most important one, 1 wash is enough for ATAC-seq) and pellet the nuclei as above.
5. Resuspend nuclei in PBS (with RNAse inhibitors) or NSB or NB, strain (if needed) to remove aggregated nuclei and debris (40 um Flowmi strainers).
6. Count the nuclei (add DAPI/Hoescht and/or PI and count on Countess).
7. For Chromium run, dilute the nuclei using PBS or NSB (RNA-seq) or NB (both RNA-seq and ATAC-seq).
Nuclei resuspended in NSB can be stored at -80.

HB - homogenisation buffer
250 mM sucrose
25 mM KCl
5 mM MgCl2
10 mM Tris-HCl, pH 8
0.1% Nonidet P40/IGEPAL

add fresh:
1 um DTT
0.4 U/ul RNAseIn
0.2 U/ul Superasin
(protease inhibitors can be added for ATAC-seq)

NSB - nuclei storage buffer
430 mM sucrose
70 mM KCl
2 mM MgCl2
10mM Tris-HCl, pH 8

add fresh:
0.4 U/ul RNAseIn
0.2 U/ul Superasin

HB and NSB buffers are filtered with 0.2 um filter.

NB – nuclei buffer
Prepare 1X dilution from 20X stock included in 10x Genomics ATAC kit.