Illumina HiSeq 4000 paired end sequencing; Endoderm Differentiation - Single Cell RNA-sequencing
Endoderm Differentiation - Single Cell RNA-sequencing
Cells  were treated with Accutase and, once obtained single cells, were filtered (40um strainers), counted and resuspended in PBS/BSA (at 1%) at 10^6/mL to sort them into 384-well plates (containing the lysis buffer and ERCCs). The hiPSC line FSPS13B was plated on vitronectin coated plates (10µg/ml, Stem Cell Technologies) and cultured in E6 media supplemented with 2ng/ml TGF-β (R&D) and 25ng/ml FGF2 making complete E8 media. Cells were maintained by weekly passaging using 0.5mM EDTA (Thermo Fisher Scientific). hiPSC colonies were treated with DMSO or 100ng/ml nocodazole for 16 hours. Following synchronisation, cells were maintained in pluripotency conditions or induced to form definitive endoderm using a 3-day protocol. On day 1 cells were cultured in CDM-PVA supplemented with 80ng/ml FGF2, 10µM LY294002, 10ng/ml BMP4, 100ng/ml Activin A and 3µM CHIR99021. On day 2 the cells were cultured in CDM-PVA supplemented with 80ng/ml FGF2, 10µM LY294002, 10ng/ml BMP4 and 100ng/ml Activin A. On day 3 the cells were cultured in RPMI-B27 media supplemented with 80ng/ml FGF2 and 100ng/ml Activin. Conversion was performed following the SMART-Seq2 protocol (https://www.nature.com/articles/nprot.2014.006) using a poly-Dt30 oligo and a TSO oligo. RNA extraction is based on the Picelli et al SS2 protocol, using a polydT primer plus the template switching oligo – TSO, as published (https://www.nature.com/articles/nprot.2014.006). Cell sorting and library preparation was carried out by the sequencing core facility at the Wellcome Sanger Institute. Single hPSCs were isolated into 384 wells plates and libraries were prepared for 120 cells/per condition using the SMART-Seq2 protocol (https://www.nature.com/articles/nprot.2014.006) and using Nextera XT DNA Library Preparation Kit – Illumina. A constant amount of spike-in RNA from the External RNA Controls Consortium (ERCC) was also added to the lysis buffer prior to sorting.
Experimental Factor: compound
dimethyl sulfoxide
Experimental Factor: sampling time point
0
Experimental Factor: single cell identifier
0hU_22829_1_382