title: GSM2836278: D10-1(2i); Mus musculus; RNA-Seq
library_construction_protocol: We collected a total of 66,000 cells from twelve time points over a period of 16 days in two different culture conditions. Single or duplicate samples were collected at day 0 (before and after Dox addition), 2, 4, 6, and 8 in Phase-1(Dox); day 9, 10, 11, 12, 16 in Phase-2(2i); and day 10, 12, 16 in Phase-2(serum). Cells were also collected from established iPSCs cell lines reprogrammed from the same MEFs, maintained either in Phase-2(2i) conditions or in Phase-2(serum) medium. For all time points, selected wells were trypsinized for 5 mins followed by inactivation of trypsin by addition of MEF medium. Cells were subsequently spun down and washed with 1X PBS supplemented with 0.1% bovine serum albumin. The cells were then passed through a 40 micron filter to remove cell debris and large clumps. Cell count was determined using Neubauer chamber hemocytometer to a final concentration of 1000 cells/ul. Single-cell RNA-Seq libraries were generated from each time point using the 10X Genomics Chromium Controller Instrument (10X Genomics, Pleasanton, CA) and ChromiumTM Single Cell 3' Reagent Kits v1 (PN-120230, PN-120231, PN-120232) according to manufacturer's instructions. Reverse transcription and sample indexing was performed using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Briefly, the suspended cells were loaded on a Chromium controller Single-Cell Instrument to first generate single-cell Gel Bead-In-Emulsions (GEMs). After breaking the GEMs, the barcoded cDNA was then purified and amplified. The amplified barcoded cDNA was fragmented, Atailed and ligated with adaptors. Finally, PCR amplification was performed to enable sample indexing and enrichment of the 3' RNA-Seq libraries. The final libraries were quantified using Thermo Fisher Qubit dsDNA HS Assay kit (Q32851) and the fragment size distribution of the libraries were determined using the Agilent 2100 BioAnalyzer High Sensitivity DNA kit (5067-4626). Pooled libraries were then sequenced using Illumina Sequencing By Synthesis (SBS) chemistry.
attributes.tag: GEO Accession
attributes.value: GSM2836278