DOI: 10.5061/dryad.70rxwdc9p
title: Single cell RNA sequencing of lateral and medial olivocochlear efferent neurons using PatchSeq

description: Olivocochlear (OC) efferent neurons are the final component of the
 descending auditory system. The lateral olivocochlear (LOC) neurons
 innervate type I spiral ganglion neurons (SGN). Their role in hearing is
 unknown, but they may have roles in protection from noise trauma or
 changing the threshold of SGN activation. The medial olivocochlear (MOC)
 efferent neurons innervate outer hair cells (OHC), inhibiting OHC function
 and thus dampening cochlear vibrations. LOC and MOC function is poorly
 understood at the single cell level, but recent use of transgenic mice
 (ChAT-IRES-Cre; tdTomato) expressing fluorescent reporters in cholinergic
 neurons, including LOC and MOC neurons, has allowed patch-clamp recordings
 from the neurons in mouse brainstem slices to begin to characterize their
 intrinsic electrical properties and synaptic activity. Recent single
 nucleus sequencing experiments from LOC and MOC neurons has given insight
 into the genetic programs that generate their function, including changes
 to expression profiles following noise trauma. Here we utilize the
 PatchSeq technique to combine patch-clamp electrophysiological recordings
 with deep single-cell transcriptome analysis from identified LOC and MOC
 neurons in transgenic ChAT-IRES-Cre; tdTomato mice. In this preliminary
 dataset, we generated patch-clamp electrophysiology data and single cell
 transcriptome data from 18 identified LOC and 13 identified MOC
 neurons. 
description: # Single cell RNA sequencing of lateral and medial olivocochlear efferent
 neurons using PatchSeq Dataset DOI:
 [10.5061/dryad.70rxwdc9p](10.5061/dryad.70rxwdc9p) We have submitted our
 gene expression data from PatchSeq experiments including a spreadsheet
 containing single-cell expression data for 18 lateral olivocochlear (LOC)
 and 13 medial olivocochlear (MOC) neurons, a report detailing expression
 profiles of individual cells, and associated patch-clamp data for a subset
 of cells. This is a standalone dataset, and is not in support of a
 corresponding manuscript. Sharing / Access information: Please contact
 [catherine.weisz@nih.gov](mailto:catherine.weisz@nih.gov). **Notes:**
 **Data and File Structure:** * “MOC_LOC_PatchSeq_expression_data.zip” .zip
 file * “LOC_MOC_PatchSeq_expression.xlsx” Excel file *
 “LOC_MOC_PatchSeq_expression_csv.csv” csv file: same as above, csv format
 * “MOC_LOC_PatchSeq_Patch_clamp_data.zip” .zip file * Patch-clamp data in
 .dat, .abf, or .txt format * “Patch_clamp_text_file_list.xlsx” Excel file
 * “PatchSeq_sample_list.xlsx”: Excel file *
 PatchSeq_Methods_Frank_Weisz_final.pdf” * “MultiQC_report”: html doc with
 expression details, supplemental file (Zenodo) **Spreadsheets:** * **LOC
 MOC PatchSeq expression:** This spreadsheet contains the single-cell
 expression data for 18 LOC and 13 MOC neurons. The first column contains
 the Ensembl gene id. Columns B-AF contain expression data for an
 individual cell, with the cell ID at the top of the column. The units are
 CountsPerMillion_Trimmed Mean of M component (CPM_TMM_Counts). Columb AG
 contains the entrezgene ID. Column AH “external_gene_name” contains the
 gene name. Column AI contains the description of the gene. * **PatchSeq
 sample list:** This spreadsheet contains the list of cells that are
 included in the expression dataset, along with details of cell type,
 recording date, animal age in post-natal days (P), for which the day of
 birth = P0, and patch-clamp file name. * **Patch clamp text file list:**
 This spreadsheet contains the recordings types and names for patch-clamp
 recordings that were collected using HEKA software and were converted to
 .txt files. **Sub-folder: “MOC LOC PatchSeq expression data”** Contains
 expression data from LOC and MOC neurons collected using the PatchSeq
 method. See “spreadsheet” section (above) for details for “**LOC MOC
 PatchSeq expression”.** Excel spreadsheets can be opened using Microsoft
 Excel, the csv version can be opened in any spreadsheet software.
 Information about gene expression metrics is explained within the
 supplementary MultiQC report file (stored on Zenodo). **Sub-folder:
 “PatchSeq Patch clamp data”** Contains patch-clamp electrophysiology data
 for a subset of the LOC and MOC neurons for which quality single cell
 expression data was available. It also includes an excel file detailing
 the recording types for each cell. These data were recorded using either
 the Molecular Devices pClamp software version 11 or the HEKA Patchmaster
 EBC10 software. Files can be opened with various software: Molecular
 devices (.abf) files can be opened with Clampfit software. This can be
 downloaded as part of the pClamp software suite (pClamp, AxoScope, and
 Clampfit; free download, does not require dongle unless the amplifier is
 being used to collect new data) at: -         
 [https://support.moleculardevices.com/s/article/Axon-pCLAMP-11-Electrophysiology-Data-Acquisition-Analysis-Software-Download-Page](https://support.moleculardevices.com/s/article/Axon-pCLAMP-11-Electrophysiology-Data-Acquisition-Analysis-Software-Download-Page) -          In addition, various converters can be used to convert .abf files to Matlab or python formats (eg. [https://www.mathworks.com/matlabcentral/fileexchange/6190-abfload](https://www.mathworks.com/matlabcentral/fileexchange/6190-abfload)) HEKA files (.dat files are the primary data files, if present the .bak, .mrk, .onl, .pul, and .sol files are companion files that automatically get opened with the associated .dat file that has the same name) can be opened with HEKA Patchmaster NEXT, Patchmaster, or HEKA Fitmaster: -          [https://www.heka.com/downloads/downloads_main.html#down_patchmaster_next](https://www.heka.com/downloads/downloads_main.html#down_patchmaster_next) -          [https://www.heka.com/downloads/downloads_main.html#down_fitmaster](https://www.heka.com/downloads/downloads_main.html#down_fitmaster) -          [https://www.heka.com/downloads/downloads_main.html#down_patchmaster](https://www.heka.com/downloads/downloads_main.html#down_patchmaster) (acquisition software) -          Full functionality requires purchase of a software dongle, but software can be run in the ‘demo’ mode to open and view data. Note: opening of some files generates a “Too many sample points” error, use the “My risk” button to clear the error and proceed. Open from within the fitmaster software, and load the .dat file. -          File converters are available for Matlab and python, for example this python converter: [https://github.com/campagnola/heka_reader](https://github.com/campagnola/heka_reader) -          In addition, .dat files were exported as .txt files for opening in any text editor or in Microsoft Excel. For these files, each column represents a ‘sweep’ which can be either a single sweep from a different voltage step in an “IV” experiment, or a single sweep to collect synaptic data. The voltage command (V) data and corresponding current response (I) data are in adjacent columns. Column titles have titles that detail the type of experiment with the numbers responding to the experiment number within the file, and the sweep number. All data from a single cell is included in the same .txt file, including IV curves, drug application, or longer single voltage steps. **File naming conventions:** Patch-clamp files are in two formats. For some cells, files were collected in pClamp and have the file naming structure that begins with the cell ID (LOC# or MOC#) followed by LT_year_day_month_file# (LT = experimenter Lester Torres Cadenas). Each set of traces is contained within a single file, and there may be multiple files per cell. All files for a given cell are in a folder. For the remainder of cells, a single file recorded using the HEKA Patchmaster software will have all recordings from a single cell. File naming convention is cell ID (LOC# or MOC#) 2Photon_year-month-day-subfile# (2Photon is the name of the rig and computer that was used for recordings, no multiphoton imaging data is presented in this dataset). The list of file names for each cell (or “none” if no patch-clamp data is included)  can be found in the attached spreadsheet “PatchSeq sample list” Files are present in the original file formats (.abf for Molecular Devices / pClamp and .dat for HEKA). HEKA files are also exported to .txt. **Variables and abbreviations:** CPM_TMM_Counts = CountsPerMillion_Trimmed Mean of M component”. This is a standard RNA-seq metric, where the number of reads is normalized to the total library size, and then further scaled to take into account large variations in the sample size factors. mV = milli volts, a unit of electrical potential in neurons pA = pico-amps, unit of electrical measurement of currents in neurons mM = milli Molar concentration Vhold = membrane holding voltage, the voltage that the internal neuron state was set at using the patch-clamp amplifier IV = current voltage, shorthand for a type of voltage-clamp experiment in which the neuron membrane potential is set to different holding potentials from -110 to ~+20 mV in 10 mV increments while recording the resulting currents in the neuron, or the equivalent protocol in current-clamp in which the neuron is held at a resting membrane potential of ~-60 mV, and the current is stepped to hyperpolarizing and depolarizing potentials by injecting current in 10 pA negative and positive steps. **Code / Software** No additional code was generated for this work.