GSM2093276: ID-002922_MGB; Mus musculus; RNA-Seq
Single-cell libraries were prepared as reported in Smartseq2 protocol (Picelli et al., 2013; Picelli et al., 2014) with some technical adaptation: 1µL of ERCC spike-Mix 1 (Life Technologies) diluted at 1:106 in RNAse-free water was added after cell sorting to the lysis buffer; template-switching oligo (TSO) was modified to add isomeric nucleotide at the 5’end to minimize background cDNA synthesis as previously described (Kapteyn et al., 2010) (5’-(iso-dC)(iso-dG)(iso-dC)AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’, Eurogentec); after first strand synthesis primer-dimers were removed using 11 µL of Ampure XP beads (Beckman Coulter). After the cDNA amplification, libraries were quantified using Qubit Hs Assay (Life Technologies) and quality of libraries were checked using Bioanalyser (Agilent) Using the modified protocol described in (Patel et al., 2014), 1 ng of cDNA was subjected to tagmentation-based protocol (Nextera XT, Illumina) using ¼ of the recommended volumes, 10 min for tagmentation at 55°C and 1 min extension time during PCR. After PCR and cDNA purification, cDNA was purified and resuspended in 15 µL of elution buffer (EB) (Qiagen). Finally, libraries were pooled (16 libraries for Miseq sequencing and sequencing was performed in paired-end mode for 2 x 75 cycles using Illumina's MiSeq.
GEO Accession
GSM2093276