GSM7731945: SI-Bone_marrow_recipient-2; Mus musculus; RNA-Seq
Small intestine was extracted from mice and Peyer's patches were removed. Then intestine was opened longitudinally, washed in PBS and cut into 0.5-1 cm pieces. The pieces were then incubated in ice cold PBS with 30 mM EDTA for 30 min and thoroughly washed in PBS. Afterwards they were cut into smaller pieces and digested three times for 10 min, 20 min and 30 min in RPMI 1640 containing 25 mM HEPES, 0.5 mg/ml Collagenase D and 10 µg/ml DNaseI. Disintegrated supernatants were collected in RPMI1640 + 10% FCS in between digestion steps. Cells were then filtered to remove clumps and Percoll density gradient centrifugation (40%/80%) was performed to purify lymphocytes. Cells were stained with antibodies against CD45, CD3 and CD4 and before sorting 7AAD was added to label dead cells. They were then sorted for CD45+CD3+CD4+7AAD- using the FACS Aria III and collected in PBS with 0.04% BSA. Prior to sorting cells were labeled with anti-CD45.1 TotalSeq-B0178 and anti-CD45.2 TotalSeqB0157 antibodies for feature barcoding. Library was prepared according to manufacturer's instructions (Chromium Next GEM Single Cell 3' Kit v3.1; 10X Genomics).