GSM3356595: RBPJ-KO_K-I-Treg_R2; Mus musculus; RNA-Seq
Spleen was isolated and target cells were sorted using FACS; RNA was isolated with the RNEasy Mini Kit cDNA was generated and amplified using 4.8 ng of total RNA (RNEeasy Mini Kit) and SMARTer Ultra Low Input RNA for Illumina Sequencing - HV (Clontech Laboratories, Inc.) according to the manufacturer's protocol. Then, sequencing libraries were prepared using the NEXT ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs) according to the manufacturer's instructions with the following modifications: The adapter-ligated double-stranded cDNA (10µl) was amplified using NEBNext Multiplex Oligos for Illumina (New England Biolabs, 25 µM primers), NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs) and 15 cycles of PCR. Final libraries were validated using Agilent 2100 Bioanalyzer (Agilent Technologies) and Qubit flourometer (Invitrogen), normalized and pooled in equimolar ratios. 50bp single-read sequencing was performed on the Illumina HiSeq 2000 v4 according to the manufacturer's protocol.