Oligo dt 15 mer primers

Total RNA was isolated from 1g of leaf tissue of Z. zerumbet by the method of Salzman et al. [13 (link)]. DNase I (Ambion, Austin, TX) treatment was carried out to remove traces of genomic DNA. For cDNA synthesis, 2μg of total RNA was used along with oligo (dT) 15-mer primers (Promega, Madison, WI), 200 units of Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega, Madison, WI) and 40 units of ribonuclease inhibitor RNasin (Promega, Madison, WI). The PDS gene was amplified from Z. zerumbet using the primers ZPDS F3 and ZPDS R3 (S1 Table). PCR was carried out using Advantage 2 Polymerase mix (Clontech Laboratories, Inc., USA) with the following conditions: 94°C for 4 min; 35 cycles of 94°C for 30sec, 58°C for 30sec, 72°C for 1min; and 72°C for 4min and then held at 4°C. The PCR product was confirmed by gel electrophoresis prior to its cloning into plasmid pGEM T easy vector (Promega, Madison, WI). The nucleotide sequence of the cloned insert was then determined using the primer pairs T Vect F and T Vect R (S2 Table) in conjunction with Big Dye Terminator Cycle Sequencing (ABI).

Cells were lysed in Trizol Reagent (Invitrogen) and total RNA extracted using the direct-zol RNA MicroPrep kit (Zymo Research). cDNA synthesis was carried out with MMLV RT enzyme (Promega, M170) using Oligo dT 15 mer primers (Promega, C1101). The amplification was done using SYBR Green JumpStart Taq ReadyMix in Applied Biosystems Quant Studio 3 Real-Time PCR System with primers listed in Supplementary Table 2. For ChiP experiments, the relative quantification procedure of the Pfaffl method was used to convert the average Ct values for each sample to relative fold-change information^{88 (link)}.

Total RNA from HeLa-ATCC and HDF-TERT cells was lysed in Trizol Reagent (Invitrogen) and extracted using the Direct-zol RNA MicroPrep kit (Zymo Research). The cDNA synthesis was carried out with MMLV RT (Promega, M170) using Oligo dT 15 mer primers (Promega, C1101). PCR amplification for XBP1 gene was performed, and products were digested with PstI and treated with T7 Endonuclease (NEB, E3321) to get rid of the hybrid XBP1s–XBP1u bands (Supplementary Fig. 1f). The DNA bands were resolved in 2% agarose gel and images were taken with GeneSnap (Syngene).