alias: GSM4546548
title: GSM4546548: SC_RNA-seq_RSet; Homo sapiens; RNA-Seq
library_construction_protocol: day 0, day 3, day 7, day 13 primed, day 13 naive, day 21 primed, day 21 naive, hiPSC primed (passage 3) and hiPSC naive (passage 3) were collected and cryopreserved. These collected samples were then subjected to FACS, for D0, D3, D7, D13 primed, D13 naive, D21 primed and D21 naive samples were sorted for PI negative, TRA-1-85 positive cells to remove dead cells and iMEF cells, while hiPSC primed (passage 3) and hiPSC naive (passage 3) samples were sorted for PI negative, TRA-1-85 positive, CD13 negative, F11R positive, TRA-1-60 positive, EPCAM positive cells to get rid of dead cells and iMEF cells as well as differentiated cells. Three samples were prepared (Fig. 1b) for subsequent library preparation, sample one contained cells isolated from D0, 3 and 7, samples two and three contained cells for primed (D13, D21, hiPSCs) and naive reprogramming (D13, D21, hiPSCs) respectively, and a small number of mixed D0, 3 and 7 cells were added to sample two and three to capture the full reprogramming trajectories and also to account for potential batch effects. The collected cells were isolated, encapsulated and library constructed using Chromium controller (10x Genomics) as per the manufacturer's instructions "Chromium Single Cell 3' Reagent Kit V2 User Guide", 10X Genomics document number CG00052 Revision 3. A total of 12 cDNA amplification cycles were used. A total of 16 cycles of library amplification were used. Sequencing was carried out using an Illumina NextSeq 500 using SBS V2 chemistry in a high-output mode according to the recommendations outlined by 10x Genomics "Chromium Single Cell 3' Reagent Kit V2 User Guide", 10x Genomics document number CG00052 Revision 3, with the exception that the second read was extended to 115b instead of 98b. Libraries were diluted according to the manufacturer's instruction "NextSeq 500 System User Guide" Illumina document number 15046563 v02 and loaded at 1.8pM. Chromium barcodes were used for demultiplexing and FASTQ files were generated from the mkfastq pipeline using the Cellranger program (v2.1.0). Alignment and UMI counting were performed to the hg19 genome as per the snRNA-seq. The same experimental procedure and the computational pipeline were also applied to generate the RSeT reprogramming scRNA-seq library shown in Extended Data Fig. 4d,e.
identifiers.namespace: GEO
identifiers.id: GSM4546548