GSM3557697: old_flowsorted_epi_1; Mus musculus; RNA-Seq
Lung tissue was perfused with sterile saline from the right to the left ventricle of the heart and subsequently inflated via a catheter in the trachea, by an enzyme mix containing dispase (50 caseinolytic units/ml), collagenase (2 mg/ml), elastase (1 mg/ml), and DNase (30 ug/ml). After tying off the trachea, the lung was removed and immediately minced to small pieces (approx. 1 mm2). The tissue was transferred into 4 ml of enzyme mix for enzymatic digestion for 30 min at 37°C.  Enzyme activity was inhibited by adding 5 ml of PBS supplemented with 10% fetal calf serum. Dissociated cells in suspension were passed through a 70 μm strainer and centrifuged at 500 x g for 5 min at 4°C. Red blood cell lysis (Thermo Fischer 00-4333-57) was done for 2 min and stopped with 10% FCS  in PBS. After another centrifugation 5 min at 500 x g (4°C) the cells were counted using a Neubauer chamber and critically assessed for single cell separation and viability. 250.000 cells were aliquoted in 2.5 mL of PBS supplemented with 0.04% of bovine serum albumin and loaded for Drop-Seq at a final concentration of 100 cells/μL. Single cell suspensions were processed through Drop-Seq to generate single-cell cDNA libraries attached to microbeads. Microbeads were counted, and amplified by PCR, and the 3' end of the cDNA prepared for sequencing. For each sample, 1 ng of pre-amplified cDNA from an estimated 1000 cells was tagmented by Nextera XT (Illumina) with a custom P5 primer (Integrated DNA Technologies). Single cell libraries were sequenced in a 100 bp paired-end run on the Illumina HiSeq4000 using 0.2 nM denatured sample and 5% PhiX spike-in. For priming of read 1, 0.5 µM Read1CustSeqB (primer sequence: GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC) was used.
GEO Accession
GSM3557697