Quantifying Tet2 Knockdown in mESCs

Tet2 knockdown efficiency was measured by quantitative PCR (qPCR) and western blot [49 (link)]. For qPCR, total RNA was isolated with an RNeasy kit (Qiagen, Chatsworth, CA, USA) and cDNA was made using SuperScript III reverse transcriptase (Invitrogen). qPCR was performed using FastStart Universal SYBR Green Master mix (Roche, Mannheim, Germany) on a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression was normalized to Gapdh. Primers used for qPCR are listed below:

Tet1 forward: GAGCCTGTTCCTCGATGTGG

Tet1 reverse: CAACCCACCTGAGGCTGTT

Tet2 forward: AACCTGGCTACTGTCATTGCTCCA

Tet2 reverse: ATGTTCTGCTGGTCTCTGTGGGAA

Gapdh forward: GTGTTCCTACCCCCAATGTGT

Gapdh reverse: ATTGTCATACCAGGAAATGAGCTT

For western blot, nuclear proteins from parental and Tet2 knock-down mESCs were extracted as previously described [61 (link)]. Nuclear protein (30 μg) was loaded on 4–12 % Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membrane. Tet2 was detected using anti-Tet2 (Abcam) antibodies. Loading control, beta-actin, was detected using anti-beta actin from Abcam.