GSM8820063: CT2F_S11; Mus musculus; RNA-Seq
Mouse larynges were collected from antibiotics treated (AB)and untreated (CT)  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). We started from the dissociation protocol by Sekiguchi & Hauser (2019) and determined the optimal dissociation condition by optimizing the enzyme composition & concentration and dissociation temperature & time. Specifically, 1 ml of modified dissociation solution contained 5 mM CaCl2, 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. After dissociation, cell suspension was filtered through 40 µm cell strainer to remove aggregates. Undigested cartilage was removed before cell straining to prevent clogging cell strainer and further impact dissociation efficiency. Dissociation was performed for each larynx separately. Single-cell suspensions from three larynges per mouse group were pooled as one sample (4 biological replicates per mouse group). 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 (10x Genomics, Pleasanton, CA). Briefly, 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). 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. Library preparation was performed to generate a single pool of cDNA containing both CT and AB samples to minimize batch effect between samples. 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 S2 flowcell (Illumina, San Diego, CA) using a 150 x 150 bp sequencing reaction targeting > 60,000 reads/cell, yielding a calculated depth of over 100K mean reads per cell for samples of the first batch and over 70K mean reads per cell for samples of the second batch. MiSeq balancing run was quality controlled using calculations based on UMI-tools (99). Sequencing of the first batch of samples was performed first to serve as a pilot experiment for the second batch.