GSM8678748: EOAD1; Homo sapiens; RNA-Seq
Tissue was transferred directly onto ice-cold sucrose buffer and homogenised by hand. The homogenate was layered on top of a sucrose cushion and nuclei were recovered after centrifugation. Nuclei were resuspended in PBS containing BSA (to prevent clumping) and RNase inhibitors, counted and diluted to 1,000 nuclei/μl. Sequencing libraries were prepared using the “single cell 3' library kit v3” with the Chromium instrument (10X Genomics). Aiming to obtain libraries for ~5,000 independent nuclei per sample. Libraries were sequenced on an Illumina NovaSeq6000 instrument (UCL Genomics), to an average of at least 100,000 reads/nucleus. Raw sequencing data were processed through the software CellRanger (10X Genomics) to assign each transcript read to its respective nucleus, we performed alignment to the transcriptome, counting reads, and processing of the unique molecular identifier tag to reduce PCR bias