GSM5025666: Experiment 2 – Time series of transplanted hESC-myogenic progenitors; Homo sapiens; RNA-Seq
Pax7-GFP+ cells were sorted by Fluorescence-Activated Cell Sorting as single cells in 96-well or 384-well capture plates using MoFlo Legacy sorter. Capture plate wells contained 5 ul of capture solution (1:500 Phusion High-Fidelity Reaction Buffer, New England Biolabs; 1:250 RnaseOUT Ribonuclease Inhibitor, Invitrogen). Single cell libraries were then prepared using the previously described SCRB-seq protocol . Briefly, cells were subjected to proteinase K treatment followed by RNA desiccation to reduce the reaction volume. RNA was subsequently reverse transcribed using a custom template-switching primer as well as a barcoded adapter primer. The customized SCRB-seq barcode primers contain a unique 6 base pair cell-specific barcode as well as a 10 base pair unique molecular identifier (UMI). Transcribed products were pooled and concentrated, with unincorporated barcode primers subsequently digested using Exonuclease I treatment. cDNA was PCR-amplified using Terra PCR Direct Polymerase (Takara Bio). Final libraries were prepared using 1ng of cDNA per library with the Nextera XT kit (Ilumina) using a custom P5 primer as previously described. Pooled libraries were sequenced on two high-output lanes of the Illumina NextSeq500 with a 16 base pair barcode read, 8 base pair i7 index read, and a 66 base pair cDNA read design. 