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Protocol for Patch-Seq of Small Interneurons.

Protocol for Patch-Seq of Small Interneurons.

STAR protocols (2020-12-31)

Marcela Lipovsek, Lorcan Browne, Matthew S Grubb

ABSTRACT

Obtaining electrophysiological recordings and gene expression information from the same neuron (Patch-seq) brings forward a unique opportunity to study the transcriptional correlates of functional properties and vice versa. Here, we provide a detailed Patch-seq protocol tailored to the specialized demands of studying small interneurons. Focusing on the technically demanding process of transitioning between patch recordings and cell extraction, our protocol describes and troubleshoots steps for successfully collecting small interneurons, allowing for multi-modal Patch-seq interrogation of this crucial cell type.

MATERIALS

Product Number

Brand

Product Description

Sigma-Aldrich

Potassium chloride, ACS reagent, 99.0-100.5%

Sigma-Aldrich

Acetic acid, glacial, ACS reagent, ≥99.7%

Sigma-Aldrich

Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture

Sigma-Aldrich

DL-2-Amino-5-phosphonopentanoic acid, solid

Sigma-Aldrich

Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M

Sigma-Aldrich

D-(+)-Glucose, ≥99.5% (GC), BioXtra

Sigma-Aldrich

Sodium bicarbonate solution, solution (7.5%), sterile-filtered, BioReagent, suitable for cell culture

Sigma-Aldrich

Adenosine 5′-triphosphate disodium salt solution, Crystalline ATP, HPLC purified, aqueous solution for RNA transcription

Sigma-Aldrich

Hydrochloric acid solution, 1.0 N, BioReagent, suitable for cell culture

Supelco

JWH-018 5-Pentanoic acid beta-D-glucuronide solution, 100 μg/mL (Acetonitrile:Water (90:10) (v/v)), certified reference material, ampule of 1 mL

Sigma-Aldrich

Sodium phosphate monobasic solution, BioUltra, 5 M in H₂O

Sigma-Aldrich

Calcium chloride solution, BioUltra, for molecular biology, ~1 M in H₂O

Sigma-Aldrich

Sucrose, BioUltra, for molecular biology, ≥99.5% (HPLC)

Sigma-Aldrich

NBQX disodium salt hydrate, ≥98% (HPLC)

Sigma-Aldrich

Sodium phosphate monobasic, ReagentPlus^®, ≥99.0%

Sigma-Aldrich

Potassium D-gluconate, ≥99% (HPLC)

Sigma-Aldrich

Sucrose, ≥99.5% (GC)

Sigma-Aldrich

Diethyl pyrocarbonate, 96% (NT)

Sigma-Aldrich

SR-95531, ≥98% (HPLC), powder

Sigma-Aldrich

Guanosine 5′-triphosphate sodium salt solution, HPLC purified, aqueous solution for RNA polymerase transcription