GSM4200054
GSM4200054: hires_d10_1 [muc9285]; Mus musculus; RNA-Seq
Lung single cell suspensions were generated in the following manner. Briefly, after euthanasia, lung tissue was perfused with sterile saline through the heart and the right lung was tied off at the main bronchus. The left lung lobe was subsequently filled with 4% paraformaldehyde for later histologic analysis. Right lung lobes were removed, minced (tissue pieces at approx. 1 mm2), and transferred for mild enzymatic digestion for 20-30 min at 37°C in an enzymatic mix containing dispase (50 caseinolytic U/ml), collagenase (2 mg/ml), elastase (1 mg/ml), and DNase (30 µg/ml). Single cells were harvested by straining the digested tissue suspension through a 40 micron mesh. After centrifugation at 300 x g for 5 minutes, single cells were taken up in 1 ml of PBS (supplemented with 10% fetal calf serum), counted and critically assessed for single cell separation and overall cell viability. For Dropseq, cells were aliquoted in PBS supplemented with 0.04% of bovine serum albumin 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 HiSeq 4000 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
GSM4200054
GEO Accession
GSM4200054