GSM6165058: ConvR, larynx, scRNAseq; Mus musculus; RNA-Seq
Mouse larynges were collected from ConvR and GF mice and minced into 0.5 mm small pieces on a sterile petri dish under a dissection scope, then digested in a 1 ml modified dissociation solution for 40 min in a cold room (4 °C). Specifically, 1 ml of modified dissociation solution contained 5 mM CaCl¬2, 90 µl Accutase (Stem Cell Technologies, Cambridge, MA), 90 µl Accumax (Stem Cell Technologies, Cambridge, MA), 10 mg/ml Bacillus lichenformis protease (MilliporeSigma, Burlington, MA), and 125 U/ml DNase I (MilliporeSigma, Burlington, MA) dissolved in sterile calcium/magnesium-free PBS. Samples were gently triturated every 5 min using a wide-bore pipette tip during dissociation. Dissociation was performed for each larynx separately. Single-cell suspensions from three larynges per mouse group were pooled together as one sample. Pooled suspensions were added to 5 ml Ca/Mg-free PBS supplemented with 10% FBS, filtered with a 40 µm cell strainer (MilliporeSigma, Burlington, MA), then centrifuged at 300 g, for 10 min, at 4°C.  Cell pellet was resuspended in 30 µl ACK lysing buffer (Lonza, Bend, OR) briefly to remove erythrocytes, following manufacture's instruction. ACK treated single cells were resuspended in 500 µl of Ca/Mg-free PBS supplemented with 2% FBS. Libraries were prepared according to Chromium Single Cell 3' Reagent Kit v3.1. single cell suspensions were transported on ice to the University of Wisconsin-Madison Biotechnology Center, where cell concentration and viability were quantified on the Countess II (Thermo Fisher Scientific, Waltham, MA) using 0.4% Trypan Blue (Invitrogen, Carlsbad, CA). Six thousand cells were added to each channel with an average recovery rate of over 4,000 cells. Cells were then partitioned into Gel Beads in Emulsion in the GemCode instrument, where cell lysis and barcoded reverse transcription of RNA occurred, followed by amplification, shearing and 5′ adaptor and sample index attachment. Libraries were quantified using the Qubit High Sensitivity DNA Kit (Thermo Fisher Scientific, Waltham, MA), uniquely indexed, pooled, balanced for the number of estimated reads per cell with a MiSeq (Illumina, San Diego, CA) sequencing run, and subsequently sequenced on a NovaSeq SP flowcell (Illumina, San Diego, CA using a 150 x 150 bp sequencing reaction targeting > 60,000 reads/cell, yielding a calculated depth of over 0.1 million reads per cell for both mouse groups. MiSeq balancing run was quality controlled using calculations based on UMI-tools101.