NextSeq 500 sequencing; RNA-seq of TET1 knockout, catalytic mutant and wild type mESCs
For RNA-seq, three different cell lines (WT J1, Tet1 KO H9, Tet1 CM D7) were cultured as independent quadruplicates. The generation of Tet1 KO (clone H9) and Tet1 CM (clone D7) mESC lines was described previously (18, 30).  Mouse ESCs were cultured in “Serum LIF” conditions and as independent replicates for 6 days prior to experiments. Here the cells were maintained on 0.2% gelatin-coated dishes in Dulbecco's modified Eagle's medium (Sigma) supplemented with 16% fetal bovine serum (FBS, Sigma), 0.1 mM ß-mercaptoethanol (Invitrogen), 2 mM L-glutamine (Sigma), 1× MEM Non-essential amino acids (Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma), LIF (ESGRO, Millipore).   RNA was isolated using the NucleoSpin Triprep Kit (Machery-Nagel) according to the manufacturer's instructions. Isolated total RNA was normalised and subjected to RNA sequencing using a version of the prime-seq method (https://www.protocols.io/view/prime-seq-s9veh66). This method is based on the single cell RNA-seq method mcSCRB-seq (90) and is a three prime counting method that includes a sample specific barcode sequence (see scan name) and unique molecular identifiers (UMI) for accurate quantification of gene expression. Here we used the Nextera XT Kit (Illumina) for sequencing library preparation as described in the mcSCRB-seq protocol (90).