GSM5129503: Control GFP rep1; Mus musculus; RNA-Seq
RNA-seq: RNA extraction was performed as detailed by manufactorer (RNeasy mini kit, Qiagen). CUT&RUN: Genomic DNA extraction was performed as per manufactorers guidelines (QIAquick Nucleotide Removal Kit). RNA-seq: Samples quantity (RNA Integrity Number = RIN) and quality were assessed using the Agilent RNA 6000 Pico Kit (Agilent #5067-1513) on the Bioanalyzer 2100. Libraries preparation started with rRNA depletion using the Ribo-Zero™ Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre for Illumina #MRZG12324). 3.0 ng to 5.1 ng of total RNA were used for rRNA depletion of the total RNA. Speed-vac sublimation was performed on some samples to reduce RNA volume to 17ul in order to start the depletion reaction. rRNA depleted RNA were then purified with AGENCOURT® RNA CLEAN® XP Kit (Beckman Coulter). The samples were eluted in 8.5ul of H2O to continue the library preparation using SMARTer® Stranded RNA-Seq Kit (Takara Bio USA, Inc. #634836 or #634837). CUT&RUN: Libraries were prepared with the KAPA DNA HyperPrep Kit (Roche 07962363001 - KK8504). This protocol includes an End-Repair/A-tailing step and an adapter ligation step followed by a PCR amplification (enrichment) of ligated fragments. RNA-seq: The 1st strand synthesis was performed using a modified N6 primer (the SMART Stranded N6 Primer) in order to obtain full length fragments. The full-length, single-stranded (ss) cDNA fragments were purified with Agencourt DNA CleanUp AMPure® XP Kit (Beckman Coulter #A63881). PCR Enrichment + indexing step of 15 cycles was then performed using the Illumina Indexing Primer Set provided by the kit. The final enriched product (library, after PCR) was purified using Agencourt DNA CleanUp AMPure® XP Kit (Beckman Coulter #A63881). Libraries were eluted in 16 ml of elution buffer. Quality and quantification of the libraries were assessed using the Agilent High Sensitivity DNA Kit (Agilent #5067-4626) on the Bioanalyzer 2100. The libraries were then quantified by q-PCR to obtain their nanomolar (nM) concentration. Libraries were diluted and pooled equimolar prior to paired-end sequencing performed with 50 cycles (PE50) on a v4 flowcell (Illumina - HiSeq PE Cluster Kit v4 cBot - PE-401-4001) of the Illumina HiSeq 2500 System. CUT&RUN: The adapters used for ligation were IDT for Illumina TruSeq UD Indexes (Illumina - 20022371). The final enriched product (library, after PCR) was purified using KAPA purification beads (Roche 07983298001 - KK8002) and a dual-SPRI size selection was performed (with KAPA beads) to select fragments between 180-500 bp. Libraries were then quantified using a Nanodrop microvolume spectrophotometer (ng/l) and quality was assessed using the Agilent High Sensitivity DNA Kit (Agilent -5067-4626) on a Bioanalyzer 2100. The libraries were then quantified by q-PCR to obtain their nanomolar (nM) concentration. Libraries were diluted, pooled equimolar and sequenced in pair end 50 cycles (PE50) on a S1 flowcell (Illumina - 20012863) of the Illumina NovaSeq 6000 System.