GSM5202089: beaf_k_rep1; Drosophila melanogaster; RNA-Seq
RNA purification was performed with RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol 3' RNA-seq library generation was performed based on a previous protocol ( Klein-Brill, A., Joseph-Strauss, D., Appleboim, A. & Friedman, N. Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex. Cell Rep. (2019). doi:10.1016/j.celrep.2018.12.020). In brief, 100ng from each sample was incubated with oligo-dT reverse transcription (RT) primers containing 7bp barcode and 8bp UMI (Unique Molecular Identifier) at 72°C for 3 minutes and moved directly to ice. This was followed by a Reverse transcription reaction (SmartScribe kit Clontech) for 1 hour at 42°C and 70°C for 15 minutes. Next, samples were pooled and purified using SPRI beads X1.2 (Agencourt AMPure XP, Beckman Coulter). The pooled barcoded samples were then tagmented using Tn5 transposase (loaded with oligos Tn5MEDS-A) for 8 min at 55°C Followed by the addition of 0.2% SDS to strip off the Tn5 and SPRI X2 purification was performed. Finally, a PCR was performed with primers containing NGS sequences (KAPA HiFi HotStart ReadyMix, Kapa Biosystems, 12 cycles) and the library was cleaned using 0.8x SPRI beads.