title: RNA sequencing of control and Gprc5c overexpression in MLL-AF9 leukemia mice
design: RNA sequencing of control and Gprc5c overexpression in MLL-AF9 leukemia mice
library_construction_protocol: 1000 GFP+ LSK cells using the following antibodies: anti-lineage [anti-CD4 (clone GK1.5), anti-CD8a (53-6.7), anti-CD11b (M1/70), anti- B220 (RA3-6B2), anti-GR1 (RB6-8C5) and anti-TER119 (Ter-119)] all PE-Cy7; anti-CD117/c-Kit (2B8)-PE; anti-Ly6a/Sca-1 (D7)-APCCy7. Sorted cells were collected into ice-cold RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen)) and stored at -80C until further usage. Total RNA isolation was using ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen) according to the manufacturerâ€™s instructions. DNAse treatment was performed using RNAse-free DNAse Set (Qiagen). Total RNA was used as starting material for cDNA-libraries preparation using the SMARTseq v4 Ultra Low RNA Kit (Takara) according to the manufacturers indications.DNAse treatment was performed using RNAse-free DNAse Set (Qiagen). Total RNA was used as starting material for cDNA-libraries preparation using the SMARTseq v4 Ultra Low RNA Kit (Takara) according to the manufacturers indications. Of note, 12 cycles were used for the amplification of cDNA. Sequencing libraries were prepared from 10ng of cDNA libraries as input using NEBNext Ultra II FS DNA library prep kit (#E7805S, NEB) according to manufacturer's protocol together with unique dual indexing barcodes (NEB, #E6440). Libraries were amplified with 7 PCR-cycles before Ampure bead (Beckman-Coultier) clean-up and subsequent quality control.