title: GSM2355299: 2i-Jaki-day2 (I); Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted using the PureLink RNA Mini Kit (Ambion, Life Technologies, Cat no. 12183018A and 12183025) according to the manufacturer’s instructions. Cells were homogenized using a QIAshredder (Qiagen, Cat no. 79654). Cell pellets were frozen at the treatment-specific time points and RNA was extracted from all pellets at the same time for each analysis. Quality of total RNA samples were checked on an Agilent Bioanalyzer 2100 RNA Nano chip following Agilent Technologies’ recommendation. Concentration was measured by Qubit RNA HS Assay ona Qubit fluorometer (ThermoFisher). RNA library preparation was performed following the NEB NEBNext Ultra Directional Library Preparation protocol. Briefly, 250 ng of total RNA was used as the input material and enriched for poly-A mRNA, fragmented into the 200-300-bases range for 4 minutes at 94oC and converted to double stranded cDNA, end-repaired and adenylated at the 3’ to create an overhang A to allow for ligation of Illumina adapters with an overhang T; library fragments were amplified under the following conditions: initial denaturation at 98oC for 10 seconds, followed by 13 cycles of 98oC for 10 seconds, 60oC for 30 seconds and 72oC for 30 seconds, and finally an extension step for 5 minutes at 72oC; at the amplification step, each sample were amplified with a different barcoded adapters to allow for multiplex sequencing. One ul of the final RNA libraries was loaded on a Bioanalyzer 2100 DNA High Sensitivity chip (Agilent Technologies) to check for size; RNA libraries were quantified by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems). Libraries were pooled in equimolar quantities and paired-end sequenced on an Illumina HiSeq 2500 platform using a High Throughput Run Mode flowcell and the V4 sequencing chemistry following Illumina’s recommended protocol to generate paired-end reads of 126-bases in length.