GSM4506250: AB5081; Mus musculus; RNA-Seq
Normal mammary fat pad: Tissue was minced using scissors and placed and disrupted for 25 min at 37oC in a gentleMACS C tube using gentleMACS dissociator, in the presence of enzymatic digestion solution containing 1 mg ml-1 collagenase II, 1 mg ml-1collagenase IV and 70 unit ml-1 DNase. Primary tumor: Tissue was minced using scissors and treated with enzymatic digestion solution containing 3 mg ml-1 collagenase A and 70 unit ml-1 DNase in RPMI 1640 for 20 min at 37oC, with pipetting every 3 min. Lung metastases: Metastases bearing lungs were harvested and placed in gentleMACS C tubes with an enzymatic digestion solution containing collagenase A 1.5 mg ml-1, dispase II 2.5 unit ml-1 and DNase I 70 unit ml-1 in RPMI 1640. The tissue was disrupted for 30 min at 37oC using gentleMACS dissociator. Following dissociation, all tissues were washed, filtered through a 70 μm cell strainer and pelleted by centrifugation at 350 g, 5min, 4°C. Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. In brief, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline. The pooled sample was linearly amplified by T7 in vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and Illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration as described previously {Jaitin, 2014}
GEO Accession
GSM4506250