Illumina HiSeq 2500 paired end sequencing; GSM2872855: 123520_NTSP3_1_A01; Mus musculus; RNA-Seq

123520_NTSP3_1_A01

Single-cell RNA sequencing has generated the first catalogs of transcriptionally defined neuronal subtypes of the brain. However, the cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of a subset of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1023 cellular responses to sensory manipulation demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type biased choices of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype. Overall design: 1023 single cell RNA-Seq and 6 bulk RNA-seq

Variation in activity state, axonal projection, and position define the transcriptional identity of individual neocortical projection neurons.

Submitted on 01-JAN-2018

For bulk samples, RNA was extracted with a Qiagen RNeasy Micro Kit. RNA quality was assessed with the Bioanalyzer Pico RNA kit and samples had RIN scores of 8.2-8.8. For bulk samples, library preparation and amplification were performed according to the Smart-Seq2 protocol (Picelli et al., 2014) with some modifications. Principally, all primer and adapter sequences were synthesized with a 5'-biotin to minimize primer dimerization. Briefly, 200 pg of RNA per sample were used as input for template switching cDNA synthesis. Full-length cDNA was amplified by KAPA HiFi mediated PCR for 20 cycles using a 5'-biotinylated ISPCR primer. Then, 500 pg of Ampure XP bead cleaned (1:1) cDNA was used as input for a standard Nextera XT tagmentation reaction, and amplification of adapter-ligated fragments was carried out for 12 cycles during which individual index sequences were added to each distinct sample. Library concentration was assessed with Qubit and library fragment size distribution was assessed on the Agilent Bioanalyzer. Pooled, indexed bulk RNA-seq samples were initially sequenced on the Illumina MiSeq 500 platform, and subsequently the same pool was sequenced on one lane of the Illumina HiSeq 2500 platform to produce 50 bp paired-end reads. Libraries were sequenced to an average depth of 4.2x107 fragments each. Reads from both runs were aggregated by index prior to mapping.  Library preparation and amplification of single-cell samples was performed using a modified version of the Smart-Seq2 protocol. Briefly, 96-well plates of single-cell lysates were thawed to 4°C, heated to 72°C for 3 minutes, and then immediately placed on ice. Template switching first-strand cDNA synthesis was performed as described above using a 5'-biotinylated TSO oligo. cDNAs were amplified using 18 cycles of KAPA HiFi PCR and 5'-biotinylated ISPCR primer. Amplified cDNA was cleaned with a 1:1 ratio of Ampure XP beads and 300 pg was used for a one-half standard-sized Nextera XT tagmentation reaction. Tagmented fragments were amplified for 12 cycles and dual indexes were added to each well to uniquely label each library. Concentrations were assessed with Quant-iT PicoGreen dsDNA Reagent (Invitrogen) and samples were diluted to ~2 nM and pooled. Pooled libraries were sequenced on the Illumina HiSeq 2500 platform to a target mean depth of ~8.0 x 105 50 bp paired end fragments per cell (Mudge and Harrow, 2015) at the Hopkins Genetics Research Core Facility to generate 50 bp paired-end reads.

Variation in activity state, axonal projection, and position define the transcriptional identity of individual neocortical projection neurons.

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