title: GSM3230898: VMP_scRNA_seq2_cell_23 dataset #3; Rattus norvegicus; RNA-Seq
library_construction_protocol: Tissues were disscociated with 2mg/mL Collagenase 1A (Sigma-Aldrich, Missouri, USA) for 60 minutes at 37 degrees C. Dispersed fibroblasts were separated by centrifugation and passing through a 0.7um cell strainer (Falcon, Corning, New York, USA). Cell suspensions were centrifuged for 10 minutes at 500g and resuspended in LIVE/DEAD Cell Viability/Cytotoxicity Assay for mammalian cells (ThermoFisher, L-3224).   After a 10 minute incubation at room temperature, cells were centrifuged and resuspended in Cell Wash Buffer (Fluidigm).  Cell concentration, size and viability were verified on hemocytometers (Incyto DHC-N01-5) through bright field, GFP and RFP on a EVOS FL Auto microscope (ThermoFisher).  Single cell RNA libraries were constructed according to the Fluidigm protocol using C1 to generate libraries for RNA sequencing (PN 100-7168). Single cell RNA libraries were constructed according to the Fluidigm protocol using C1 to generate libraries for RNA sequencing (PN 100-7168).  Briefly, full length mRNA-Seq libraries were generated from single-cells captured on the Fluidigm C1 platform using SMARTer Ultra Low RNA Kit (P/N 634936 Clontech). ERCC RNA Spike-In mix (P/N 4456740 ThermoFisher) was added to the lysis mix for normalization and quality control purposes. Full length cDNAs were converted into sequence ready libraries using Nextera XT DNA Sample Preparation Kit (P/N FC-131-1096 Illumina), and sequenced on an Illumina HiSeq 2000 with paired-end 100bp read option. The Fluidigm C1 platform captured 52 and 70 single cells from VMP and SU respectively. The average full length cDNA yield/min/max were 6.06ng(+/-0.12)/2.25ng/14.81ng for VMP and 8.51ng(+/-0.21)/2.55ng/26.6ng for SU.  Libraries from 52 VMP and 63 SU single cells were sequenced. Lysates were clarified from sonicated nuclei and AR-DNA complexes were isolated with antibody. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end)
attributes.tag: GEO Accession
attributes.value: GSM3230898