GSM8243860: 3263; Mus musculus; RNA-Seq
Wild-type mice were anesthetized by isoflurane after which they were sacrificed by cervical dislocation. The GI tract was harvested with care to prevent nicking major blood vessels. The mesenteric fat was removed as much as possible. The colonic bowel contents were flushed out with ice-cold 1X PBS (which did not contain RNase inhibitor or Actinomycin D). After cleaning the tissue was transferred to a petri-dish containing ice-cold Opti-MEM (with 2uM Actinomycin D). The LM-MP tissue was peeled on a sterile 1 ml pipette and placed in sterile RNase free tubes containing maintenance media (composition given below) and kept on ice. After all the animals have been sacrificed and LM-MP isolated, using new single edged blade, the LM-MP was chopped into minute pieces. The chopped tissues were digested in 50ml conical tubes with digestion media (composition given below) for 35 mins at 37C with shaking. The tubes were then vortexed for 30 sec at the highest speed followed by trituration with 1ml pipette tips. Samples were centrifuged for 7mins at 200g at temperature of 4C. Supernatants were aspirated gently and re-suspended in 4-5ml of Maintenance Buffer. The re-suspended pellets were passed through a 100 micron cell sieve after which the eluents were triturated with fire polished glass pipettes. The samples were centrifuged for 7mins at 200g, the supernatants aspirated, and the pellets resuspended in 3-4ml of Maintenance Buffer followed by trituration with glass pipettes. The washing process was repeated once more tand the pellets finally re-suspended in 1ml Maintenance Buffer and filtered through 40micron cell sieve and submitted to Hopkins Single Cell Sequencing Core. Upon submission, the quality of the samples (viability and cell count) was assessed with Trypan Blue and Cell Countess (Invitrogen). The library was made using Chromium Next GEM Single Cell 3' Kit v3.1 (10X genomics). The depth of reads was 20,000/cell and the sequencing was carried out by NovaSeq 6000 (Illumina).