Illumina HiSeq 4000 paired end sequencing; RNA-seq of Pluripotent Stem Cell-Derived Endometrial Stromal Fibroblasts in Monolayer Differentiation and in Co-culture with Endometrial Epithelial Organoids
RNA-seq of Pluripotent Stem Cell-Derived Endometrial Stromal Fibroblasts in Monolayer Differentiation and in Co-culture with Endometrial Epithelial Organoids
RNAeasy Minikit (Qiagen # 74104) was used to isolated RNA from ESC differentiated cells for sequencing. Monolayer cells were dissociated to single cell suspension using Accutase and pelleted. RNA was isolated following manufacturers protocol. pLOC-TurboRFP (Horizon) used to transduce constitutive RFP expression in PSC derived cells. The virus was added to adherent cultures with 4ug/mL of polybrene (EMD Millipore #TR-1003-G) in differentiation media. The next day, media containing virus was removed and replaced with fresh differentiation media. The day after, cells were dissociated to a single-cell suspension to be sorted by FACS. RFP+ cells were collected, pelleted, and RNA was isolated following manufacturers protocol. RNA quality was accessed by a Bioanalyzer 2100 (Agilent technology) with a RIN value cutoff of 6.9. No dissection was performed Monolayer Cell Culture and Differentiation: Both embryonic (H7) and iPS (reprogrammed from Coriell fibroblast GM03651 by Sendai virus (ThermoFisher #A16517) following manufacturer's instruction) cells lines of female origin (XX) were used for differentiation as well as co-culture.  Cells were cultured in mTeSR1 media (STEMCELL Technologies #85875) and on Corning Matrigel hESC-Qualified Matrix (Corning™ #354277) coated tissue culture plastic plates. Lot specific dilution factor volume provided by the manufacturer is diluted into 24mL of cold DMEM/F12 (ThermoFisher Scientific # 11320-082). To coat: 1mL of this solution is applied to 1 well of a 6 well plate (9.6cm2) and incubated at RT for 1hr or 37C for 30min. To differentiate, PSC were dissociated to single-cell level using Accutase (EMD Millipore # SCR005). Once at single cell level, equal volume mTeSR1 was added to stop dissociation and cells were pelleted at 0.2e3 rcf for 5 min. Supernatant was discarded and the remaining pellet of PSC was resuspended in mTeSR1 and 10mM Y27632 (Reprocell #04-0012-02) and plated onto Corning Matrigel hESC-Qualified Matrix coated tissue culture plates at 3x105 per 3.8cm2 or equivalent density. Media is replaced with Differentiation Basal Media (Advanced RPMI 1640 (Gibco #12633020), 1x Gluatmax (Gibco #35050061) and 8mM CHIR99021 (STEMCELL Technologies #72054). After 4 days, the media is changed to Differentiation Basal Media containing 100ng/mL recombinant Human WNT7A (R&D Systems # 3008-WN-025). The final differentiation treatment media was Differentiation Basal Media with 10ng/mL Recombinant Human BMP2 (R&D Systems # 355-BM-010), and 10nM b-estradiol (E2, Millipore Sigma #E2758-250MG). While it varies by cell line, the expected yield at day 12 off differentiation when starting with 3x105 stem cells is 0.6x106 – 13.2x106 PSC-ESF.  To passage, differentiated cells were dissociated using Accutase and pelleted at 0.2x103 rcf for 5 min. The supernatant was discarded, and the remaining pellet of PSC was resuspended in Differentiation Basal Media at a density of 3x106 per 2cm2 on Corning Matrigel hESC-  Qualified Matrix coated plates. Cells can be maintained in Differentiation Basal Media for up to 3 passages for monolayer decidualization. For decidualization treatment, cells are pretreated with 10nM E2 in DMEM/F12, no phenol red (ThermoFisher Scientific #21041025), 1x Glutamax, and 1x ITS-A (ThermoFisher Scientific #51300044) for 1 day. Then media is changed to contain 2% charcoal stripped FBS (ThermoFisher Scientific #12676029). Decidualization treatment media contained 0.5mM 8-Br-cAMP (Tocris #1140), 1mM Medroxyprogesterone 17-acetate (Millipore Sigma #M1629), and 10nM E2. Vehicle media contained equal volume of EtOH (Millipore Sigma #E7023) and Cell Culture Grade Water (Corning # 25055CVC). RNAeasy Minikit (Qiagen # 74104) was used to isolated RNA from ESC differentiated cells for sequencing Libraries were generated by TruSeq stranded total RNA library prep kit (Illumina) and TruSeq RNA CD Index Plate.