title: GSM7734998: cell_36; Mus musculus; RNA-Seq
library_construction_protocol: Preparation of RNase-free internal solution and cleaning of the equipment was performed as described (Cadwell et al., 2017). Coronal brainstem slices were prepared (Hirtz et al., 2011) and in vitro electrophysiological recordings were performed at 36.5  1 °C (Müller et al., 2019). The internal solution contained 0.8 % RNase inhibitor . Electrophysiological characterization was limited to 10-15 min to minimize mRNA degradation; thereafter, total RNA was extracted into the patch pipette by aspirating the neuron's cytoplasm via gentle suction. Shrinkage of the soma and entering of the nucleus into the pipette were monitored visually under DIC optics (Suppl. Fig. 1 A1 - A3). Upon successful harvesting, atmospheric pressure was reinstalled, and the pipette was slowly removed from the soma (Suppl. Fig. 1 B). In case of contamination by extracellular material on the pipette, absence of a membrane patch at the pipette tip or soma swelling, samples were discarded (Suppl. Fig. 1 C1, C2, D1, D2). The content of the pipette was collected in a reaction tube containing 2 μl lysis buffer . Samples were stored at -80 °C and processed further en bloc (library preparation and sequencing). From a total of 263 patched neurons, 103 remained for further analysis (ultimately, 86 neurons surpassed RNA quality control, see below). One negative control was collected per experimental day. To do so, a pipette filled with internal solution was moved to touch the slice with its tip. It was then retracted, and the pipette's content was processed as for harvested cells. Libraries were generated using a modified Version of Smartseq2 followed by Illumina Nextera XT library preparation. RNA-seq (Patch-seq)