GSM2977673: ETX-A-Side1; Mus musculus; RNA-Seq
To collect RNA for RNA-Seq, ETX-embryos generated using a T/Brachyury-GFP reporter line were collected and TSC compartments were dissected away. The ESC compartment was further dissected in half on the long side and each half was analysed separately under a fluorescent microscope to identify GFP+ve (prospective posterior) and GFP-ve (prospective anterior) cells. Samples were collected in lysis buffer (2.3μl of 0.2 % Triton X-100 (Sigma) supplemented with 1U/μl RNAsIN (Ambion)) and stored at -80 C until library preparation. Low-input RNA-seq was performed using the Smart-seq2 protocol using 18 cycles of amplification in the PCR-preamplification step. A pool of indexed libraries was sequenced on a HiSeq 2500 in rapid run mode. Reads were mapped to the M. musculus genome (Ensembl version 38.77) and quantified using the HTSeq-count.