GSM9203547: Multiome, D6 Z2KO AntNPCs, ATAC; Mus musculus; ATAC-seq
Nuclei isolation from WT and Z2KO D3 progenitor cells and D6 AntNPCs was performed by washing cells twice with PBS before detaching them from the tissue culture plate with Stempro™ Accutase™ Cell Dissociation Reagent (Thermo Scientific, A1110501). The Accutase incubation times were adjusted to the cell type (6 min for D3 progenitors and 10 min for D6 AntNPCs). Next, Accutase was inactivated with BSA 0.04% in PBS. Cell clumps were disrupted by gently pipetting up and down the cell suspension in the collection tubes. Cell suspension was then centrifuged for 5 min at 300 rcf. The supernatant was aspirated without disrupting the cell pellet and cells were washed twice in BSA 0.04% in PBS. Then, cells were diluted 1/10 in BSA 0.04% in PBS and counted. Next, 106 cells were transferred to a DNA LoBind® 1.5 ml microtube (Eppendorf, 0030108051). Cells were centrifuged again at 300 rcf for 5 min at 4ºC. Next, the supernatant was carefully aspirated and cells were lysed by adding 175 µl of pre-chilled Lysis Buffer (10X Genomics) to the pellet while smoothly pipetting up and down 10 times. After incubation for 4 min on ice, suspension was centrifuged at 500 rcf for 5 min at 4º C. Then, supernatant was completely removed without disrupting the nuclei pellet. Nuclei suspension was cleaned up by adding Wash Buffer (10X Genomics) followed by centrifugation (500 rcf, 5 min at 4ºC). Assuming ~50% nuclei loss during cell lysis, cells were resuspended in chilled Diluted Nuclei Buffer (10X Genomics) to achieve the desired final nuclei concentration. The rest of the steps of the scATAC-seq and scRNA-seq coupled protocol were performed exactly as indicated in the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide CG000338 Rev F. Single-cell 3′ gene expression libraries were prepared using the Chromium Next GEM Multiome ATAC + Gene Expression kit (10x Genomics) according to the manufacturer's protocol. Briefly, nuclei were isolated and loaded into the Chromium Next GEM Chip J, where individual nuclei were partitioned into Gel Bead-In-Emulsions (GEMs) together with a barcoded oligonucleotide gel bead and reverse transcription reagents. Within each GEM, mRNA transcripts were reverse transcribed to generate barcoded cDNA containing both a unique cell barcode and a unique molecular identifier (UMI). After breaking the emulsions, cDNA was recovered, amplified by PCR, and purified. Final 3′ gene expression libraries were constructed through enzymatic fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR, followed by quality control and quantification. ATAC libraries were prepared from the same nuclei using the Chromium Next GEM Multiome ATAC + Gene Expression kit (10x Genomics) following the manufacturer's protocol. Isolated nuclei were transposed with Tn5 transposase, which inserted sequencing adapters at accessible chromatin sites. Transposed DNA was then loaded into the Chromium Next GEM Chip J with barcoded gel beads and amplification reagents. In GEMs, DNA fragments were amplified with primers containing a cell barcode and UMI, enabling single-nucleus resolution. Following GEM breakage, libraries underwent additional amplification and enrichment PCR steps to incorporate sequencing adapters and sample indices. Libraries were purified, quality-checked, and quantified prior to sequencing.