GSM8393316: cre_atac_rep1; Mus musculus; ATAC-seq
We extracted nuclei from live tdTomato+ cells sorted from spheres by FACS, using the nucleus extraction buffer (10mM Tris pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween 20, 0.1% NP40, 0.01% Digitonin and 1% BSA). Prior to nuclei loading on the microfluidic chip, we adjusted the nuclei concentration for each sample to fall within the recommended concentration range provided by the manufacturer. Using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle (10x Genomics, 1000285), Following transposition and GEM generation, we performed post GEM- cleanup and library pre-amplification PCR. Subsequently we proceeded with ATAC library and gene expression library construction, performed QC on the generated libraries and submitted them for sequencing. Gene expression libraries were pooled with cell multiplexing libraries and sequenced on the NovaSeq 6000 (Illumina, San Diego, CA) to generate a minimum 2,000 reads per cell for cell multiplexing libraries and 20,000 reads per cell for gene expression libraries in a 28/10/10/90 (R1/i7/i5/R2) bp configuration according to 10X Genomics guidelines. ATAC-seq libraries were pooled and sequenced on NovaSeq 6000 (Illumina, San Diego, CA) to generate 25,000 reads per cell for 10,000 targeting cells, equivalent to ~250 million paired-end reads per library in a 50/8/24/50 bp configuration according to 10X Genomics guidelines.