GSM8137801: Aged-Flox; Mus musculus; RNA-Seq
Small intestines were removed and the fat/mesentery was dissected away. The intestinal lumen was washed with ice cold PBS (Mg2-Ca2-) using a 18G feeding needle (Roboz FN- 7905) until the intestines appeared white/pink. They were then opened longitudinally. The mucous was removed by gently rubbing the intestine between fingers in cold PBS. The intestines were cut into 3–5-mm fragments and placed into 50-ml conical tubes that were filled with ice-cold 30 ml of PBS (Mg2-Ca2-/)EDTA (10 mM). The samples were incubated and shook intermittently on ice for 30 min continuously discarding and replacing (at least three times) the supernatant. The fragments were then continually re-suspended with ice-cold 30 ml PBS (Mg2-Ca2-)EDTA (10mM) and intermittently shook on ice for 10 min, discarding the supernatant again for three times. The fragments were re-suspended again with ice-cold 30 ml PBS (Mg2-Ca2-)/EDTA (10mM) and incubated and intermittently shook while waiting on ice for 30 min. The samples were then triturated with a 10-ml pipette 1 to 2 times, and the contents were filtered twice through a 70- m mesh (BD Falcon) into a 50-ml conical tube to remove villous material and tissue fragments. The tissue suspensions were centrifuged for 5 min at 300g (4 C or room temperature), and the pellets were gently re-suspended in 1.0 ml of undiluted TrypLE Express (Invitrogen), 120 l of DNase I (10U/l, Roche) and transferred to 15-ml conical tubes. The samples were incubated in a 32 C water bath for 3 min (placed 15ml tubes inside water filled 50 ml tube on 32 C heat block), were not titurated, and were then placed on ice. Twelve millilitres of cold SMEM was added to each sample and were gently triturated twice. The samples were then centrifuged for 5 min at 300g.   The pellets were re-suspended and incubated for 15 min on ice in 0.5–1 ml SMEM that contained an antibody cocktail consisting of CD45-PE (eBioscience, 30-F11), CD31-PE (Biolegend, Mec13.3), Ter119-PE (Biolegend, Ter119), CD24-Pacific Blue (Biolegend, M1/69) and EPCAM-APC (eBioscience, G8.8).  Use 10l of each antibody. Do not add 7AAD to unstained samples. 15 ml tubes: No GFP unstained, PE antibodies only. CD24 Pac. Blue only, EPCAM only, 7AAD only, GFP unstained, 7AAD with all stains GFP. Twelve millilitres of SMEM were added and the samples were centrifuged for 5 min at 250g. The pellets were resuspended with 0.5–2 ml (depending on the size of the pellet) of SMEM/7-AAD (7-aminoactinomycin D) solution (1:500 dilution). 150l of 7-AAD in 1~2ml of MEM medium. Stained samples were filtered through a 40- m mesh/ filter (FLOW tubes) before cell sorting. Single cell libraries were prepared on a 10x Genomics Chromium controller (10x Genomics, Pleasanton, CA) using the version 3 reagent kit according to the manufacturer's instructions. The reagent mixture was calculated for capturing 6000 cells. The libraries were sequenced on a NovaSeq (Illumina, San Diego, CA) by the NIEHS Epigenomics core laboratory. Raw read processing was carried out using the Cell Ranger Single-Cell Software Suite (version 3.0.1, 10X Genomics).