title: GSM7853048: Multiome_ATAC_E13.5pax9KO; Mus musculus; ATAC-seq
library_construction_protocol: Samples were mechanically broken into single-nuclei suspensions following the manufacturer's instructions for Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing (10X Genomics—protocolCG000375). After isolation, the nuclei integrity was accessed under a microscope and counted using the CellDrop (Denovix) and acridine orange (AO) and propidium iodide (PI) as dye. Transposition and Gel Bead-in-Emulsion (GEM) generation, using a Chromium Single Cell Multiome ATAC + 3′ Gene Expression following the manufacturer's instructions (10x Genomics—protocolCG000338Rev F) were performed, aiming for a total of 10,000 nuclei per sample. In brief, the nuclei were transposed in a bulk solution using Tn5 transposase with sequencing adapters that were pre-loaded. The transposed nuclei were loaded onto a microfluidic chip to create GEMs. The gel beads contained two types of oligos that were used for snRNA-seq and snATAC-seq. The mRNA was reverse transcribed and underwent template switching and transcript extension to produce barcoded cDNA. The snATAC-seq oligo was formed of a partial Illumina primer sequence, a 10X barcode, and a spacer that allowed the barcode to attach to the adapter-tagged DNA fragments. The GEMs were broken using a recovery agent to produce a bulk pool of barcoded molecules. The pre-amplified product was then split into portions available for snRNA-seq and snATAC-seq library construction. P5, i7, and P7 sequences compatible with Illumina bridge amplification were added to the DNA fragments through PCR to produce snATAC-seq libraries. Additional amplification of the cDNA was performed on the snRNA-seq samples before proceeding to fragmentation, end repair, A-tailing, and ligation. A sample dual index PCR was used to add the P5, i5, i7, and P7 sequences to the final snRNA-seq library. Multiome: RNA-Seq and ATAC-Seq