GSM5983934: Mouse #63 Lym; Mus musculus; RNA-Seq
For RNA isolation, the Qiagen RNeasy Micro Kit (Qiagen, Hilden, Germany) was used according to the manufacturer's protocol. Library generation:   2ng of total RNA (except for sample N2591: 1ng) were used for library preparation with the 'SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian' (#634413; Takara/Clontech) according to conditions recommended in user manual #063017. Generated libraries were barcoded by dual indexing approach and were finally amplified with 12 cycles of pcr. Fragment length distribution of generated libraries was monitored using 'Bioanalyzer High Sensitivity DNA Assay' (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the 'Qubit® dsDNA HS Assay Kit' (Q32854; ThermoFisher Scientific).    Sequencing run:   Equal molar amounts of nine libraries were pooled along with additional unrelated libraries for a common sequencing run. Each of the nine analyzed libraries constitutes 10% of overall flowcell capacity. The library pool was denatured with NaOH and was finally diluted to 2pM according to the Denature and Dilute Libraries Guide (Document # 15048776 v02; Illumina). 1.3 ml of denatured pool was loaded on an Illumina NextSeq 550 sequencer using a High Output Flowcell for single reads (20024906; Illumina). Sequencing was performed with the following settings: Sequence reads 1 and 2 with 38 bases each; Index reads 1 and 2 with 8 bases each.