GSM9537348: 2xMBD2 EM-seq nuc rep1; Homo sapiens; OTHER
Preparation of single-cell suspensions: Human brain organoids were dissociated into single-cell suspensions as previously described using the Neural Tissue Dissociation Kit (P) (Milternyi Neural Dissociation protocol). Organoids were cut into pieces using a scalpel, thoroughly washed in DPBS supplemented with 0.5% BSA, and incubated with papain-containing Enzyme Mix 1 at 37 °C for 15 min, followed by DNase-containing Enzyme Mix 2. Mechanical dissociation was performed by sequential trituration using P1000 and P200 pipette tips with intermittent 37 °C incubations until a homogeneous suspension was obtained. Cells were filtered through a 30 μm strainer, pelleted at 300 × g for 5 min, resuspended in wash buffer, and counted using Trypan Blue. Following dissociation of organoids or tissues, genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, #69504) according to the manufacturer's instructions. Following dissociation of organoids or tissues, genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, #69504) according to the manufacturer's instructions. CUT& Tag and DNA-Methylation CUT&Tag: Starting with 0.1-1 Mio cells after dissociation, cells were transferred into CUT&Tag wash buffer (20 mM HEPES [pH 7.5] (Jena Bioscience, #CSS-511), 150 mM NaCl (Sigma Aldrich, #S6546), 0.5 mM Spermidine (Sigma Aldrich, #S0266), 5 mM sodium butyrate (Sigma Aldrich, #303410), Roche Protease Inhibitor (Sigma Aldrich, #11873580001)). Following 15 µl of BioMag ConcanavalinA beads (Polysciences, #86057-3) in binding buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) were added to the sample and incubated on the wheel for 15 min at RT. Subsequently, the cells were collected on a magnet and lysed through the addition of CUT&Tag wash buffer supplemented with 0.01% Digitonin. Lysis was monitored under a microscope with Trypan Blue staining. After lysis was complete, the nuclei were washed again with CUT&Tag wash buffer. If possible, all samples were split, and H3 or another chromatin mark CUT&Tag was performed on the same starting material, to be used as a normalizer. The antibody (1 µg per reaction, against histone modifications or 5mC) was added together with 2 mM EDTA final and the sample was incubated on a rocking platform at 4 °C overnight. For DNA methylation profiling, MBD-Tn5 was added for 2 hours in a buffer containing 300 mM salt. ProteinA coupled Tn5 (pA-Tn5) was used a control and treated in the same way. The next day, the samples were washed once with CUT&Tag wash buffer, and the secondary antibody was added to the reaction, which was then incubated for 1 hour at 20 °C on a rocking platform. For CUT&Tag After two additional washes the Tn5 was added (1:100) in CUT&Tag med buffer (20 mM HEPES [pH 7.5] (Jena Bioscience, #CSS-511), 300 mM NaCl (Sigma Aldrich, #S6546), 0.5 mM Spermidine (Sigma Aldrich, #S0266), 5 mM sodium butyrate (Sigma Aldrich, #303410), Roche Protease Inhibitor (Sigma Aldrich, #11873580001)). Tn5 was allowed to bind for 1 hr at 20 °C on a rocking platform. After two additional washes, the cutting was induced through the addition of 10 mM MgCl2 in CUT&Tag med buffer. For DNA methylation profiling, the MBD-Tn5 cutting was directly induced after the 2-hour binding and two washes with CUT&Tag med buffer. For DNA methylation profiling of purified DNA, 50 ng of starting material was used, and MBD-Tn5 cutting was directly induced after a 2-hour binding period without any additional washes. After 1 hr at 37 °C, the reaction was stopped by adding a final concentration of 20 mM EDTA, 0.5% SDS, and 10 mg Proteinase K. The reaction was then incubated at 55 °C for 30 min and finally inactivated at 70 °C for 20 min. The DNA fragments were purified using the ChIP DNA Clean & Concentrator kit (Zymo Research, #D5205). For elution from the columns, 2 pg of Tn5-digested and purified lambda DNA (New England Biolabs, #N3011S) were added as a spike-in normalizer for later analysis. Overview of the antibodies used in the study: 5mC Diagenode C1520003 H3K27ac Diagenode C15410196 H3K36me3 Abcam AB9050 H3K4me1 Diagenode C15410194 H3K4me3 Diagenode C15410003 H3K4me9 abcam ab176916