Transcriptomics

This Technical White Paper describes the transcriptomics profiling of the dorsal part of the lateral geniculate complex (LGd). Part of the initial goal of the Allen Institute’s cell type project is to characterize, in a systematic and standardized manner, individual neurons in the LGd and primary visual cortex (VISp or V1) of the young adult laboratory mouse. Descriptions of the tissue preparation, RNA amplification and library preparation for RNA-Seq, RNA-Seq data processing, and clustering are provided. Slices were prepared from P53-P59 mice and were sectioned (250 μm) using a vibrating microtome. Each slice was imaged to aid in brain region identification and registration to the Allen Mouse Common Coordinate Framework (CCF). For more information on the CCF please see the whitepaper in the Documentation tab. Regions of interest were microdissected under a fluorescence dissecting microscope. Dissected tissue pieces were treated with protease and subsequently triturated. Single cells were then isolated by fluorescence-activated cell sorting (FACS). cDNA amplification and library construction were performed using SMART-Seq v4 (Clontech) and Nextera XT (Illumina) kits. Single cell libraries were sequenced on HiSeq (Illumina) to generate 50 base-pair paired-end reads. The reads were aligned and after alignment, quality control was performed and two independent methods were utilized to identify sets of clusters.