title: GSM7031204: HuEmCtxfresh40mm10-5w_201029_S8; Homo sapiens; RNA-Seq
library_construction_protocol: The nuclei isolation from the embryonic brain tissue was performed according to Jönsson et al., 2021. In brief, the tissue was thawed and dissociated in ice-cold lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) using a 1 ml tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose cushion (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 min in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before being resuspended in 300 ml of dilution buffer (10 mM Tris-HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C at a low flow rate using a 100 mm nozzle (reanalysis showed >99% purity). Single cell/nuclei: Nuclei/cells were directly loaded onto the Chromium Next GEM Chip G or Chromium Next GEM Chip K Single Cell Kit along with the reverse transcription mastermix following the manufacturer's protocol for the Chromium Next GEM single cell 3' kit (10X Genomics, PN-1000268) to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics using 13 cycles of amplification for 3' libraries. Bulk RNAseq: Total RNA was isolated from nuclei or tissue using the RNeasy Mini Kit (Qiagen). CUT&RUN: We followed the protocol detailed by the Henikoff lab (Skene et al., 2017). Briefly, 100,000 sorted nuclei were washed twice (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x Roche cOmplete protease inhibitors) and attached to 10 ConA-coated magnetic beads (Bangs Laboratories) that had been pre-activated in binding buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Bead-bound cells were resuspended in 50 μL buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 0.5 mM Spermidine, 1x Roche complete protease inhibitors, 0.02% w/v digitonin, 2 mM EDTA) containing primary antibody (rabbit anti-H3K4me3 Active Motif 39159, RRID:AB_2615077; or goat anti-rabbit IgG, Abcam ab97047, RRID:AB_10681025) at 1:50 dilution and incubated at 4°C overnight with gentle shaking. Beads were washed thoroughly with digitonin buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete protease inhibitors, 0.02% digitonin). After the final wash, pA-MNase (a generous gift from Steve Henikoff) was added to the digitonin buffer and incubated with the cells at 4°C for 1 h. Bead-bound cells were washed twice, resuspended in 100 μL digitonin buffer, and chilled to 0-2°C. Genome cleavage was stimulated by the addition of 2 mM CaCl2 at 0°C for 30 min. The reaction was quenched by the addition of 100 μL 2x stop buffer (0.35 M NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 50 ng/μL glycogen, 50 ng/μL RNase A, 10 fg/μL yeast spike-in DNA (a generous gift from Steve Henikoff)) and vortexing. After 10 min of incubation at 37°C to release genomic fragments, cells and beads were pelleted by centrifugation (16,000 g, 5 min, 4°C) and fragments from the supernatant were purified. Single cell/nuclei: Sequencing libraries were generated with unique dual indices (TT set A) and pooled for sequencing on a Novaseq6000 using a 100-cycle kit and 28-10-10-90 reads. Bulk RNAseq: Libraries were generated using Illumina TruSeq Stranded mRNA library prep kit (poly-A selection) and sequenced on a NextSeq500 (PE 2×150bp). CUT&RUN: Illumina sequencing libraries were prepared using the Hyperprep kit (KAPA) with unique dual-indexed adapters (KAPA), pooled and sequenced on a Nextseq500 instrument (Illumina).