title: GSM4732439: h1hESC_EC-EQ-Vary_13; Homo sapiens; RNA-Seq
library_construction_protocol: Single-cell suspensions were loaded onto a medium size (10-17 μm) C1 Single-Cell Auto Prep IFC (Fluidigm), and cell-loading script was performed according to the manufacturer's instructions. Full-length single-cell cDNA libraries generated by the SMARTer PCR cDNA Synthesis kit (Clontech) and the Advantage 2 PCR kit (Clontech) performed in the C1 system. For EQ, EQ-Vary and EQ-75%, we retrieved the harvested cDNA, which are amplified full-length single-cell cDNAs identical to those in GSE75748. For the EQ and EQ-75% experiments, 2.0 µL of the amplified/tagmented cDNA were used for pooling. In the EQ-Vary experiment, a single scaling factor was applied to generate variable amounts of the pooling volume. These pooled single-cell libraries were used in an AMpure XP Bead-based Dual Bead Cleanup and Size Selection reaction (Agencourt AMPure XP PCR Purification modified Instructions for Use, Beckman Coulter). In both bead cleanup reactions, 90% of AMPure XP beads were added to the amplified single-cell libraries to select for an approximate size range of 150-700 bp and incubated for 15 minutes at room temperature. Libraries bound to beads were then placed on a magnet for 5 minutes, washed twice with 70% Ethanol, eluted with Suspension Buffer (Nextera XT DNA Sample Preparation Index Kit, Illumina), and transferred to a new tube.  Final amplified and pooled single-cell libraries were quantified with the Qubit dsDNA HS Assay Kit (Q32854, Thermofisher) and Bioanalyzer High Sensitivity DNA Analysis Kit (5067-4626, Agilent).Libraries were multiplexed with 96 samples per lane and sequenced on an Illumina HiSeq3000 with paired-end 65 or 78 bp reads. Single-cell RNA-Seq