Protocols: A total of 14 RtOgs on Day 57 (D57) and 12 RtOgs on D171 were dissociated using papain-based enzymatic digestion (Worthington papain dissociation NJ, USA). Cell viability was tested with 0.4% trypan blue in a hemocytometer (>90%). The concentration was adjusted to ~870 live cells/μl for day 171 samples and ~822 cells/μl for day 57 ones. The samples were prepared for scRNA-seq library within 5 minutes. The resulting gel-in-emulsions (GEMs) were transferred to PCR tubes and incubated in a Bio-Rad C1000 Thermocycler (Bio-Rad Laboratories, Hercules, CA) for the reverse transcription protocol. The GEMs were cleaned up using Dynabeads MyOne SILANE (Life Technologies, Carlsbad CA) and then amplified using 11 cycles according the 10X workflow. The cDNA was cleaned using 0.6X SPRIselect (Beckman Coulter, Indianapolis, IN) size selection and then quality control assays using Qubit DNA HS assay (Life Technologies, Carlsbad CA) and Agilent 2100 Bioanalyzer DNA HS (Agilent, Santa Clara, CA) were performed. The endogenous cDNA fraction was then processed according to the 10X workflow for library construction. The cDNA was fragmented, end repaired and then A-tailed. After a SPRIselect cleanup the adapters were ligated on the cDNA. Sample indexes were added by PCR and a double-sided size selection using SPRIselect was performed.
ENA first public
2022-01-01
ENA last update
2022-01-01
External Id
SAMEA10774180
INSDC center alias
UNIVERSITY OF CALIFORNIA, IRVINE
INSDC center name
UNIVERSITY OF CALIFORNIA, IRVINE
INSDC first public
2022-01-01T00:13:15Z
INSDC last update
2022-01-01T00:13:15Z
INSDC status
public
Submitter Id
E-MTAB-11121:Retinal Organoids Day 57
broker name
ArrayExpress
cell line
CSC14
cell type
induced retinal cell
common name
human
growth condition
organoid culture
progenitor cell type
embryonic stem cell
sample name
E-MTAB-11121:Retinal Organoids Day 57