GSM4826964: PPF.P30.0.A; Mus musculus; RNA-Seq
RNA-seq and scRNA-seq: For mouse fibroblasts, fibroblasts were isolated from fresh specimens using FACS. RNA extraction was performed using Qiagen miRNeasy kit (cat. 1071023) with on column DAnase treatment per the manufacturer's recommendations. The Clontech Smarter Ultra Low Input RNA kit (Takara Bio, Cat. 634848) was used to generate cDNA from 150 pg total RNA following the manufacturer's recommendations.  Amplified cDNA was purified using SPRI Ampure Beads (Beckman Coulter, Cat. A63880) and the quality and quantity was measured using a High Sensitivity DNA chip on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA-seq: The Smarter Ultra Low Input RNA kit, from TakaraBio formerly Clontech (Catalog# 634848) was used to generate cDNA from total RNA using the standard protocol.  Amplified cDNA was purified using SPRI Ampure Beads from Beckman Coulter (Catalog # A63880). The quality and quantity was assed using a High Sensitivity DNA chip on the Agilent 2100 Bioanalyzer.  Illumina Nextra-XT kit (Catalog #FC-131-1024) was used to generate libraries from 150pg of amplified cDNA following standard protocol.  Equal nanomole of uniquely indexed libraries was pooled and sequenced on HighSeq4000. (2X75 pair-end). scRNA-seq: cDNA was sheared to an average length of 300 basepairs using a Covaris S2 ultrasonicator (Covaris) and libraries were generated with the Clontech Low Input Library Prep kit (Takara Bio, Cat. 634947).  scRNA-seq libraries were prepared using the 10x Genomics Chromium platform. scRNA-seq: The samples were uniquely barcoded, pooled, and sequenced onto two lanes of the NextSeq 500 (Illumina). A total of 300 million paired-end, 151 base pair reads were obtained, resulting in 50 million reads per sample.