The details of the recipe can be found below. The FBS must be filtered under sterile conditions. Before use, the complete growth medium must be warmed up in a bottle bath (37°C). The bottle must be stored at 4°C for up to 6-months. Growth Medium ReagentFinal concentration (vol %)...
Cells were then lysed with Cell lysis buffer (Bioke) for 10 min at room temperature (RT) and fluorescence relative to 100% cell death was measured and used as normalization parameter. For other cell death assays, cells were collected 24 h after treatment with the cell death inducers ...
A custom sequencing recipe was used for dark-cycling during the first 18 bases, covering the flanking primer region, with the read output starting at the beginning of the barcode and extending 10 bp into the other flanking priming region. BarSeq gene fitness calculations BarSeq reads were ...
Single stranded DNA oligo labels (hashes) were then added to nuclei (aiming for approximately 0.5 pmol hash molecules per 1000 cells) in lysis buffer and incubated on ice for 5 min. Hashes have the sequence 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXBAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3...
Finally, organoids were transferred to Neural Maturation Media (Ndiff+VA, same recipe as Ndiff-VA but with B27 supplement containing Vitamin A) on day 6 and media was changed every 2-3 days until day 14. Matrigel was added to the corresponding media at increasing dilutions during organoid ...
Tagmentation plates were prepared by the combination of 1430 μl of TBS with 770 μl nuclei solution. The TBS recipe was described in “Blocking barcode-swapping”, but a different version of Digitonin (Bivision 2082–1) was used here. This solution was mixed briefly on ice; 20 μl of ...
Whole cell lysates were obtained by lysing the cells on ice in NP-40 lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 1 mM DTT). Cytoplasmic and nuclear fractions were isolated using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, ...
The cross-linked cells were sonicated in lysis buffer (50 mM HEPES–NaOH, pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% Na-deoxycholate, 1% TritonX-100 and 0.1% SDS) to shear the chromatin DNA into 200- to 400-bp lengths. Chromatin was immunoprecipitated with primary antibodies over...
The basic recipe of C-minimal medium is as below: 16 g/L K2HPO4, 4 g/L KH2PO4, 2.32 mg/L MnSO4·4H2O, 0.123 g/L MgSO4·7H2O, 12.5 µM ZnCl2, 50 mg/L tryptophan, and 22 mg/L ferric ammonium citrate. 20 mM NH4Cl was used as the nitrogen source....
Probes are then neutralized by the addition of 5 μL 10% acetic acid and ethanol precipitated [L. Angerer, "In situ hybridization with RNA probes, and annotated recipe", in Applications to Neurobiology, Oxford University Press, Oxford UK (1986)]. For each experimental condition, 5 to 10×...