Pbr322 vector

Absolute telomere length was determined according to published protocol (O'Callaghan & Fenech, 2011). DNA from spleen and liver was isolated using DNeasy Blood and Tissue kit (Qiagen) with slight modification of adding 1 mm DTT to eluate. Briefly, a standard curve of known quantities of a synthesized 84 mer oligonucleotide containing only TTAGGG repeats was generated based on CT (cycle threshold) using iQ SYBR Green Supermix (Bio‐Rad). A standard curve using the single copy gene, 36B4, was generated to serve as a control for amplification and to determine genome copies per sample. For both standard curves using synthesized oligomers, 20 ng of plasmid DNA (pBR322 Vector, New England Biolabs) was loaded in all wells. For samples, 20 ng of DNA was loaded in triplicate. Synthesized oligomers and primer sequences for qPCR in Table S2 and all samples were processed on a 96CFX real‐time system (Bio‐Rad).

To study the effects of GATA3 on CRC cells, we first constructed recombinant plasmids of GATA3-OE and GATA3-shRNA. The GATA3-shRNA sense sequence was 5′-CACCGGACGAGAAAGAGTGCCTCAATCAAGAGTTGAGGCACTCTTTCTCGTCCTTTTTTG-3′ (the underlined part is the target sequence, and “TCAAGAG” is the stem-loop structure). The annealed double-stranded DNA was ligated into the pGpU6/GFP/Neo vector (P05464, miaolingbio, China). The overexpressed GATA3 vector was used to amplify GATA3 by PCR. The sequence of the primers was as follows: forward, 5′-GCCTCTGCTTCATGGATCCC-3′, and reverse, 5′-CTGAGATTCCAGGGGGAGGC-3′. The amplified target gene was ligated with pBR322 vector (N3033L, NEB, USA) in the presence of restriction enzymes Hpa I/EcoRI/Xho I (TAKARA, China) and T4 DNA ligase (20325, TRANS, China). After the plasmid sequencing and identification was completed, plasmids were extracted from the fresh bacterial solution amplified from the cloned colonies using the plasmid large extraction kit (CW2104, Kangwei, China).