Spri beads

P4 anterior cerebellums were dissected and washed with cold 1x PBS, then homogenized in cold ATAC lysis buffer (10 mM HEPES, pH 7.4, 10 mM NaCl, 1.5 mM MgCl2, 0.1% IGEPAL CA-630) with tight Dounce. Nuclei were filtered with a 40 µm cell strainer. In all, 50,000–100,000 nuclei were washed with 500 µL of cold ATAC lysis buffer then pelleted by gentle centrifugation. Nuclear pellets were resuspended in 50 µL of transposition reaction mix (25 μL of 2x TD Buffer (Illumina, FC-121-1030), 5 µL of Tn5 Transposase (Illumina, FC-121-1030), 5 µL of 0.1% Digitonin, 5 µL of 0.2% Tween-20 and 10 µL of H₂O). The transposition reaction mix is incubated at 37 °C for 30 min in a thermomixer set at 800–1000 rpm. DNA was purified with Qiagen MinElute PCR kit and amplified using 10 cycles of PCR reaction (25 µL of DNA, 5 µL of 10 µM Custom Adapter Mix i5 and i7, 10 µL of 5x Reaction Buffer, 1.5 µL of 10 mM dNTPs, 0.5 µL of Q5 Polymerase (NEB), 8 µL of H2O). The amplified DNA was purified with Beckman Coulter SPRI beads and sequenced on the Illumina NextSeq 500 platform to obtain 40 bp paired-end reads (Center for Genome Sciences at Washington University). Three biological replicates for ATAC-seq experiments.

Following dissociation, cells were diluted to 110 cells/µl in PBS/0.01% BSA. Drop-seq was performed as described in Macosko et al.^{79 (link)}. Briefly cells and barcoded beads (Chemgenes, 132 beads/µl in lysis buffer) were run on an aquapel-treated microfluidic drop-seq device (FlowJEM) for co-encapsulation in nanoliter-sized droplets. After droplet-breakage, reverse transcription and Exonuclease I treatment, beads were counted, and 2500 beads were apportioned per PCR tube for cDNA amplification. The amplified cDNA libraries were purified using SPRI beads (Beckman Coulter) and quantified on a Fragment Analyzer (Agilent). Tagmentations were performed using the Illumina Nextera XT kit (Illumina), and the resulting libraries were purified in two consecutive rounds of SPRI beads-based size selection (0.6x beads to sample ratio followed by 1x beads to sample ratio). The size and concentration of the final libraries were measured on a Fragment Analyzer and a Qubit Fluorometer (Thermo Fisher), respectively. Libraries were sequenced on an Illumina Nextseq500 instrument.