GSM5548425: Enthesis Gli1 P42; Mus musculus; RNA-Seq
Tendon entheses from P11, P18, and P56 C57BL/6J mice were dissected under surgical microscope and pooled together. They were digested in HBSS solution with 300 mg/ml liberase, 20 ng/ml Dnase, 5% FBS, and 100 mg/ml HEPES for 1.5 hours at 37 celsius degree. The samples were filtered through a 40 μm strain, resuspended in the RBC lysis buffer for 5 minutes at room temperature and stained with Cd45 in 200 μl PBS with 5% FBS for 45 minutes at 4°C. Then, the samples were washed with PBS three times, resuspended in 500 μl PBS with 1 ug/ml DAPI and 5% FBS, and filtered into polypropylene FACS tubes. Single cells without Cd45 and DAPI staining were index sorted by a BD influx Sorter and collected into a microcentrifuge tube for scRNA-seq 10 Genomics experiments. For dissociation of Gli1-lineage cells, tendon enthesesof GliCreERT;Ai14 mice of P7, P11, P18, P21, P28, and P42 were dissected, pooled together, and digested in the aforementioned solution under the same condition discussed above. All cells were stained with 1 ug/ml DAPI and were index sorted by a BD influx Sorter. Single cells without DAPI staining and red fluorescence were collected into 96-well plates, prefilled with lysis buffer for scRNA-Seq analysis. For all enthesis cells, resulting pooled, 3'-end libraries was sequenced on Illumina® NovaSeq™ 6000; For Gli1-lineage cells, Pooled 3'-end sequencing libraries were prepared and finally sequenced on an IIlumina NextSeq 500