| description |
We performed mRNA-seq. Our samples are induced pluripotent stem cell (iPSC:201B7), which proportion of total colonies were controlled by morphological analysis and colony pickup/elimination. To mimic the slightly different culture conditions resulting from differences in human skills even when using the same protocol, we set the proportion of colonies with irregular morphologies to either increase or decrease using a combination of morphology analysis and colony pickup. First, when a certain number of colonies were obtained in 6-well plates during the preparation culture of iPSCs, phase contrast images were acquired. Acquired images were processed using image analysis software with colony recognition recipes. Two conditions showing altered morphological variation were set; Bad morphology area minimized condition (BM_min), and Bad morphology area enhanced condition (BM_enhance). To prepare either condition in a well, cellular objects outside the field of view (1.6 cm2 in the center position of a well) were scraped off using a scraper. Next, the entire cellular area recognized by colony recognition recipe was scraped off with a pipette and scraper. The remaining cells were then picked up for seeding into new 6-well plate using scraper. This condition was designated as BM_min. In contrast, the cellular area recognized by GM_recipe was removed in the same manner and the remaining cells were picked up for seeding into new 6-well plate in the same manner, designated as BM_enhance.
This manipulation day was counted as day 0. At day 4, when cell growth was in the logarithmic growth stage, the cellular objects outside the field of view were again scraped off to eliminate the cellular existence outside the analyzed images. Next, poly-dimethylpolysiloxane (PDMS) was inserted to fill the space except for the field of view. In the well containing the PDMS insert, the recognition and quantification of cellular area was analyzed.
As a result the coverage ratio of bad morphology recognized by image analysis software were 0.2% (BM_min-1), 0.3% (BM_min-2), 20.8% for (BM_enhance-1), and 22.8% (BM_enhance-2). By measuring the lactate production rate per cell (LacR) and the glucose consumption rate per cell (GLuR), the contrast value of LacR/GluR was calculated to select 2 wells in each BM_min or BM_enhance. From the selected 2-wells from BM_min or BM_enhance, total mRNAs were extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.) and RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturers instructions. The integrity of rRNA in each sample was checked using an Agilent TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA). The library was prepared for conventional RNA-seq using a commercial kit (TruSeq RNA Sample Kit; Illumina, San Diego, CA, USA) in accordance with the manufacturers protocol. Sequencing of the libraries using conventional RNA-seq was carried out using NextSeq 500 (Illumina) according to the manufacturers protocol. |