GSM8862664: Enteric neuro-glial culture; IL-13 + IFNg (24h); replicate 9; Mus musculus; RNA-Seq
Newborn P0 mouse pups were euthanized and the small intestine with mesentery was isolated, cleaned, and placed in holding buffer—i.e., HBSS without Ca2+ and Mg2+ (Gibco), 5% FCS, 10mM HEPES (Gibco), 100 units mL-1 Pen-strep (Gibco), 100 μg mL-1 Gentamycin (Lonza)—until all tissues were collected. Small intestines were then minced with scissors and digested for 30 min at 37C with magnetic stirring at 550 rpm in digestion buffer—i.e., HBSS with Ca2+ and Mg2+ (Gibco), 5% FCS, 10mM HEPES (Gibco), 100 μg mL-1 DNase I (Roche), 200 μg mL-1 Liberase TH (Roche). After digestion, samples were gently triturated with 2-3 passes through an 18G needle, filtered with a 40μm strainer, and quenched by washing 3-times with ice-cold sterile DMEM/F-12 media (Gibco). Cells from each mouse were then plated in distinct wells of a 12-well low-adhesion plate and incubated at 37C with 5-10% CO2 for 6-7 days in Neurosphere Culture Media—i.e., DMEM/F-12 (Gibco), 10mM HEPES (Gibco), 100 units mL-1 Pen-strep (Gibco), 100 μg mL-1 Gentamycin (Lonza), 2mM Glutamine (Gibco), 55μM β-mercaptoethanol (Gibco), 1x B-27 Plus supplement (Thermo Scientific), 1x N2 supplement (Thermo Scientific), 20 ng mL-1 recombinant FGF-basic (PeproTech), 20 ng mL-1 recombinant EGF (PeproTech), 50 ng mL-1 recombinant GDNF (R&D Systems). Debris and dead cells were then gently removed with 2-3 media washes, centrifuging at 80 x g for 1-2 min to enrich the precipitate for neurospheres. Neurospheres were dissociated in Accutase (Stem Cell Technologies) for 15-20 min with gentle trituration. After counting cells, dissociated neurospheres were plated in 96-well flat-bottom plates (10,000-20,000 cells per well) or 8-well imaging slide chambers (Ibidi; 20,000-40,000 cells per well) coated with Matrigel (BD; 1:100 dilution in DMEM/F-12 for 2 hrs at 37C) and incubated at 37C with 5-10% CO2 in Neuron Differentiation Media—i.e., Neurobasal A medium (Gibco), 10mM HEPES (Gibco), 100 units mL-1 Pen-strep (Gibco), 100 μg mL-1 Gentamycin (Lonza), 2mM Glutamine (Gibco), 55μM β-mercaptoethanol (Gibco), 1x B-27 Plus supplement (Thermo Scientific), 1x N2 supplement (Thermo Scientific), 50 ng mL-1 recombinant GDNF (R&D Systems). Experiments with cytokine and drug incubations began after day 10 and visible inspection of neuronal networks in culture. After the conclusion of experiment, wells were washed once gently with PBS and RNA from cultures was isolated for bulk RNA sequencing or RT-qPCR by the addition of 5-7 μL of Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma-Aldrich), snap-frozen on dry ice, and stored at -80C until further processing. A modified version of the SMART-Seq2 protocol (Picelli, et al. Nat Protoc. 2014.) was applied to generate full-length transcriptomic data from low-input samples—processed by the Klarman Cell Observatory and the Broad Institute Genomics Platform (GP). Briefly, RNA from lysates were cleaned-up using Agencourt RNAClean XP SPRI magnetic beads (Beckman Coulter, A63987) and first strand cDNA synthesis was performed using Maxima H-minus Reverse Transcriptase (Thermo Fisher Scientific, EP0753) and amplified using KAPA HiFi HotStart PCR ReadyMix (KAPA Biosystems, KK2602). cDNA concentration was assessed using Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) and subsequently normalized to 0.25 ng μL-1. Library preparation was performed using the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096), confirmed to have a size of 500 bp using a BioAnalyzer (Agilent), and pooled libraries were normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flow cell cluster amplification and sequencing were performed according to the manufacturer's protocols using the paired-end Illumina sequencing (2 x 38 bp) with 75 cycle Nextseq 500 high output V2 kit (Illumina).