GSM1963975: BC07LN_36; Homo sapiens; RNA-Seq
Dead cells in single cell suspensions were removed by Ficoll-Paque TM PLUS (GE healthcare) separation before loading to integrated fluidic circuit (IFC) chip. Each suspension was loaded to 10-17um IFC for mRNA sequencing chip then cDNA synthesis and amplification were performed with SMARTer Ultra Low RNA Kit (Clontech) in the C1TM Single-Cell Auto Prep System (Fluidigm). RNA spike-ins 1, 4 and 7 from ArrayControl TM RNA Spikes (ThermoFisher) were added to the lysis mix for validation of array and batch effect. Bulk RNAs were extracted from ~1x10^5 cells of suspensions or tumor tissues using RNeasy Plus Micro kit (Qiagen) and 10ng of total RNAs were amplified with SMARTer kit (Clonetech) under the same conditions as single cells. All of amplified cDNAs were quantified and qualified by the Qubit® 2.0 Fluorometer (Life Technologies) and 2100 Bioanalyzer (Agilent Technologies). Total 549 single cell cDNAs and 14 bulk samples were subjected to the RNA sequencing. Starting with 0.375 ng of amplified cDNAs, sequencing libraries were constructed with the Nextera XT DNA Sample Prep Kit (Illumina). All of constructed libraries were quantified and qualified by the Qubit® 2.0 Fluorometer (Life Technologies) and 2100 Bioanalyzer (Agilent Technologies) then pooled without index overlapping. Finally pooled libraries were sequenced using the HiSeq2500 (Illumina) as 100-bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit.
GEO Accession
GSM1963975