GSM9064847: HTO file: CAF23 MC, DT28 SC, DT28 MC, CAF23/DT28 CC, T47DCO SC, T47DCO MC, CAF23/T47DCO CC; Homo sapiens; RNA-Seq
To remove any cells in the dishes that had not formed part of a cluster, the cells were filtered over a 15 µm Pluristrainer (Pluriselect 43-50015-50) and then inverted to release the clusters. Clusters were resuspended in PBS and then digested with Accutase to create a single cell suspension. Cells were washed again with PBS in LoBind tubes (Eppendorf) and resuspended in PBS. Single cell preparations (grown under adherent conditions) were also made for each cell line and washed in PBS. Samples of single cells, mono-clusters and co-clusters were also made with the estrogen receptor positive cell line, T47DCO and their sequences are included in the file sets. However, we did not include analysis of T47DCO in the published paper. Each sample was incubated with a separate Hashtag Oligo antibody (Supplemental Table 1); two different experiments were performed at different times and therefore HTO10 was used for two different samples. HTO stained BC and CAF cells from each sample were given to the University of Minnesota Genomics Center to be made into 10x Chromium 3'GEX cDNA libraries (10X Genomics) following the manufacturer's protocol. In the frist experiment, El-Ashry 001, 4 samples were pooled. In the second experiment, El-Ashry 002,  8 samples were pooled. Briefly, cell suspension, gel beads, and paritiioning oil were loaded into the Chromium Next GEM chip and run through the Chromium controller to produce GEMS which are then run through a reverse-transcription reaction.  After this step, beads were cleaned up and gene expressin and HTO cDNA libraries were made. during cDNA library production, enzymatic fragmentation, end repair, A-tailing, adaptor ligation, and PCR are performed. Illumina During library construction Illumina the P5 and P7 primers used for Illumina sequencing were added. cDNAs were first evaluated by a MiSeq run and then all libraries were run on a single NovaSeq S4 gel.