title: GSM2429110: RNAseq_2i_Rep1; Mus musculus; RNA-Seq
library_construction_protocol: RNA isolation was performed using 8 time points: Day1, Day3 and Day7 of serum-2i and 2i-serum transitions and both steady-states (serum and 2i) samples. RNA isolation was performed using Qiagen RNeasy protocol and 4 microgram RNA was employed for generating the RNA-seq libraries. Total RNA was subjected to rRNA depletion using the Ribo-ZeroTM Gold kit (Epicentre) according to manufacture instruction. rRNA-depleted samples were employed in first and second strand cDNA synthesis and by using dUTP for strand-specificity. Sequencing libraries were then prepared using Kapa-Hifi (KAPA Biosystems) according to manufactory’s instruction and the final libraries were sequenced on HiSeq 2000 Illumina sequencer. Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C.Subsequently adapters were  ligated  by adding 30µl ligation buffer, 10 Kapa l DNA  ligase, 5µl diluted adaptor  in a total volume of 110µl and incubated for 15 minutes at 15°C.Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and 300bp fragments selected using E-gel. Correct size selection was confirmed by BioAnalyzer analysis.   Sequencing was performed using Illumina HiSeq 2000 machines and generated 43bp single end reads.
attributes.tag: GEO Accession
attributes.value: GSM2429110