title: GSM4761352: CD249pos Fibroblasts/mouse 7; Mus musculus; RNA-Seq
library_construction_protocol: Qiagen Rnease Micro kit: as per manual The SMART-Seq V4 Ultra Low RNA Input Kit for Sequencing (Takara Bio USA, Inc., Mountain View, CA) was used for reverse transcription and generation of double stranded cDNA for library preparation using the Nextera XT Library Preparation kit (Illumina, San Diego, CA).  Total RNA quality was analyzed via the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and RNA quantified using QubitTM (ThermoFisher Scientific, Waltham, MA) fluorometric quantitation.  An input of 10 ng total RNA was used for oligo(dT)-primed reverse transcription, cDNA amplification, and cDNA cleanup.  The cDNA was analyzed on the 4200 TapeStation (Agilent Technologies).  Quantification of cDNA was performed using Qubit.  cDNA normalized to 30 pg/ul was fragmented and sequencing primers added simultaneously.  A limiting-cycle PCR added Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for cluster formation on the sequencing flow cell.  Indexed libraries were cleaned up, library size verification performed on the 4200 TapeStation, and library quantified via Qubit.  Libraries were sequenced on a NextSeq 500 using with a 1x75 bp read length and coverage of ~50M reads/cell.