alias: GSM2047397
title: GSM2047397: NA19098-r1-G03; Homo sapiens; RNA-Seq
library_construction_protocol: Single cell loading and capture was performed following the Fluidigm manual "Using C1 to Generate Single-Cell cDNA Libraries for mRNA Sequencing Protocol" (PN 100-7168). Briefly, 30 ul of C1 Suspension Reagent was added to a 70-ul aliquot of ~17,500 cells. Five ul of this cell mix were loaded onto 10-17 um C1 Single-Cell Auto Prep IFC microfluidic chip (Fluidigm), and the chip was then processed on a C1 instrument using the cell-loading script according to the manufacturer's instructions. Using the standard staining script, the iPSCs were stained with StainAlive TRA-1-60 Antibody (Stemgent, PN 09-0068). The capture efficiency and TRA-1-60 staining were then inspected using the EVOS FL Cell Imaging System (ThermoFisher)(supplemental Table X). Immediately after imaging, reverse transcription and cDNA amplification were performed in the C1 system using the SMARTer PCR cDNA Synthesis kit (Clontech) and the Advantage 2 PCR kit (Clontech) according to the instructions in the Fluidigm user manual with minor changes to incorporate UMI labeling (Islam et al. 2014). Specifically, the reverse transcription primer and the 1:50,000 Ambion® ERCC Spike-In Mix1 (Life Tech) were added to the lysis buffer, and the template-switching oligos which contain the UMI (5-bp random sequence) were included in the reverse transcription mix. When the run finished, full-length, amplified, single-cell cDNA libraries were harvested in a total of approximately 13 ul C1 Harvesting Reagent and quantified using DNA High Sensitivity LabChip (Caliper). A bulk sample, a 40 ul aliquot of ~10,000 cells, was collected in parallel with each C1 chip using the same reaction mixes following the C1 protocol of "Tube Controls with Purified RNA" (PN 100-7168, Appendix A). For sequencing library preparation, fragmentation and isolation of 5' fragments were performed according to the UMI protocol (Islam et al. 2014). Instead of using commercial available Tn5 transposase, Tn5 protein stock was freshly purified in house using the IMPACT system (pTXB1, NEB) following the protocol previously described (Picelli et al. 2014). The activity of Tn5 was tested and shown to be comparable with the EZ-Tn5-Transposase (Epicentre). Importantly, all the libraries in this study were generated using the same batch of Tn5 protein purification. For each of the bulk samples, two libraries were generated using two different indices in order to get sufficient material. All of the 18 bulk libraries were then pooled and labelled as the "bulk" for sequencing.
identifiers.namespace: GEO
identifiers.id: GSM2047397