cycle_1_veh-diff2
Protocols: 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.
ENA first public
2021-05-15
ENA last update
2021-05-15
External Id
SAMEA8615038
INSDC center alias
UNIVERSITY OF CHICAGO
INSDC center name
UNIVERSITY OF CHICAGO
INSDC first public
2021-05-15T00:06:56Z
INSDC last update
2021-05-15T00:06:56Z
INSDC status
public
Submitter Id
E-MTAB-10375:cycle_1_veh-diff2
batch
2
broker name
ArrayExpress
cell line
H9
cell type
derived endometrial stromal fibroblast
common name
human
cycle
1
day of differentiation
12
decidualization replicate
2
developmental stage
embryo
differentiation number
5
disease
normal
genotype
wild type genotype
growth condition
co-culture with organoid
organism part
inner cell mass
originates from
day_12-batch2-diff5
progenitor cell type
embryonic stem cell
sample name
E-MTAB-10375:cycle_1_veh-diff2
sex
female