GSM9168972: Mouse E7.0 gastrulation GEX; Mus musculus; RNA-Seq
Nuclei isolation was performed using a modified 10x Genomics low cell input protocol. 45-70K cells per embryo were washed in cold PBS/0.04% BSA, then lysed in chilled lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonident P40 substitute, 0.01% digitonin, 1% BSA, 1 mM DTT, 1U/µl RNase inhibitor) for 3 min on ice. Nuclei were washed in wash buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20, 1mM DTT, 1U/µl RNase inhibitor), pelleted, and resuspended in diluted nuclei buffer. >95% nuclei viability was confirmed by trypan blue staining. Single nuclei were loaded on Chromium Next GEM Chip J (10x Genomics) for GEM generation. ATAC and gene expression libraries were constructed separately following 10x Genomics Multiome manufacturer's instructions. Library quality was assessed using Agilent D1000/D5000 ScreenTape and quantified with Qubit 4 Fluorometer. Sequencing was performed on Illumina NovaSeq 6000 in paired-end mode with the following settings: ATAC library (read1: 50bp, index1: 8bp, index2: 24bp, read2: 49bp); RNA library (read1: 28bp, index1: 10bp, index2: 10bp, read2: 90bp).