GSM8889301: M2_CM_wt_2; Mus musculus; RNA-Seq
Quant Seq 3' mRNA-seq Library Prep kit (Lexogen) was used for library construction by oligodT priming. The primer already contained Illumina-compatible linker sequences. After first strand synthesis the RNA was removed and second strand synthesis was initiated by random priming and a DNA polymerase. The random primer also contained Illumina-compatible linker sequences. Second strand synthesis was followed by a magnetic bead-based purification step. The library was then amplified, introducing the sequences required for cluster generation. External barcodes were introduced during the PCR amplification step. Library quantification was performed by fluorometer (Qubit) and Bioanalyzer (Agilent). Sequencing was performed on NextSeq500 ILLUMINA instrument to produce at least 10 million of reads (75bp SE) for sample.  QuantSeq Forward contained the Read 1 linker sequence in the second strand synthesis primer, hence NGS reads were generated towards the poly(A) tail and directly corresponded to the mRNA sequence. QuantSeq FWD maintained strand-specificity and allowed mapping of reads to their corresponding strand on the genome, enabling the discovery and quantification of antisense transcripts and overlapping genes.