GSM8796069: FL_Shox2trac_E145_DECOM_REP2; Mus musculus; RNA-Seq
Forelimbs and Hindlimbs sorted cells and entire forelimbs cell suspession were centrifuged for 5' at 400g at 4°C, supernatant was discarded and cells frozen at -80°C. Total RNA was isolated using the RNeasy Micro Kit (QIAGEN, ID:74004) following manufacturer's instructions and then stored frozen at -80°C. Total RNA was quantified with a Qubit (fluorimeter from Life Technologies) and RNA integrity assessed with a Bioanalyzer (Agilent Technologies). The SMART-Seq v4 kit from Clontech, (#634893) was used for the reverse transcription and cDNA amplification according to manufacturer's specifications, starting with 5 ng of total RNA as input. 200 pg of cDNA were used for library preparation using the Nextera XT kit from Illumina, FC-131-1096. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies). Libraries were pooled at 2 nM and loaded for clustering on a Single-read Illumina Flow cell for an average of 35 mio reads / library. Reads of 50 bases were generated using the TruSeq SBS chemistry on an Illumina HiSeq 4000 or Illumina NovaSeq 6000 sequencer.