Trizol reagent method

Exponential cell cultures (1–3 × 10⁵ cells/ml) of the different Tetrahymena species were harvested by centrifugation at 2800 rpm for 3 min. Total DNA was isolated using the protocol described in [73 (link)] and samples were treated with 10 mg/ml RNase A (Thermo- Scientific) for 2 h at 37 °C. Total RNA samples were isolated from previously stressed exponential cell cultures using the TRIzol Reagent method (Invitrogen). RNA samples were treated with DNase I (Roche) for 30 min at 37 °C. DNA and RNA integrity was tested by agarose gel electrophoresis. MultiScribe Reverse Transcriptase 50 units/μl (Life Technologies) and oligo(dT)-adaptor primer (Roche) were used to synthesize the cDNAs from 3.5 μg of the total RNA samples.

Total RNA from untransfected HeLa and HEK293 cells, human skin fibroblast and mouse brain or cerebellum was isolated using TRIZOL Reagent method (Invitrogen). The generation of cDNA from 5 μg RNA template was carried out with First-Strand cDNA Synthesis Kit (Amersham Bioscience). Human cDNA from cortex, cerebellum and liver were purchased from ClonTech Laboratories, Inc. 0ne μl of the first-strand mixture was added to 50 μl of the PCR mix containing 200 μM of dNTPs and 10 μM of sense and antisense primers (see Table 1). Amplified products were resolved on a 2% agarose-gel, purified using a QIAquick Gel Extraction kit (QIAGEN) and subjected to fluorescent DNA sequencing. All analyses were done at least in duplicate and reproduced.Table 1

Sequence of the primers used to perform the PCR for the different fragments of ataxin-2 and GAPDH as loading control

RT-PCR	Primer name	Sequence 5’to 3´	Length (bp)
A	A-Forward	TGGGCAGAGGTCGAAACAGTAACA	24
	A-Reverse	TGCATCCCAGGGCTCCAGGTC	21
B	B-Forward	AATGTTCAGACTTTGTTGTGGTA	23
	B-Reverse	TCGGGTTGAAATCTGAAGTGTGAG	24
C	C-Forward	TGAGGGGCACAGCATAAACACT	22
	C-Reverse	CGTAGGAGATGCAGCTGGAATAGG	24
D	D-Forward	GGGGGAACGTGGTCATCAGTGGT	23
	D-Reverse	GGTTGCACGCCTGGGCTC	18
E	E-Forward	TCAGCCAAAGCCTTCTACTACCC	23
	E-Reverse	CATGTTGGCTTTGCTGCTGTC	21
F	F-Forward	CCCAAATTACCATACAACAAGGAG	24
	F-Reverse	GATGTGTTCATGACTTTCAAGG	22
GAPDH	Forward	TTCACCACCATGGAGAAGGC	20
	Reverse	GGCATCGACTGTGGTCATGA	20

Plants carrying the pPLDZ2‐GUS::GFP and 3XM‐EZ2‐GUS::GFP were grown for 7 dag in Pi‐sufficient medium and then transferred to liquid medium without Pi for 12, 24, 48, 72 and 96 h, or liquid medium with Phi (1 mm); plants were then collected, frozen and ground to isolate total RNA using the TRIzol reagent method (Invitrogen). The same lines were grown in Pi‐limiting medium and then transferred to Pi‐sufficient liquid medium for 0.5, 1, 3, 6 and 12 h. cDNA was synthesized with SuperScript III reverse transcriptase (Invitrogen) using 30 μg of total RNA for each sample. The qPCR was performed in a Real‐time PCR ABI PRISM 7500 sequence detection system (Applied Biosystems), using SYBR Green PCR Master Mix (Applied Biosystems) and specific primers (Table S2). The PCR conditions were as follows: 10 min at 95 °C and 40 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 40 s. Relative transcript abundance was determined using the ACTIN2 transcript as control. At least three independent PCRs were performed for each sample. The same procedure for qRT‐PCR assay was used to analyse plants carrying the 3XM‐EZ2Bi grown in medium P+ (1 mm) for 7 dag and transferred for 72 h to P− (0 mm) liquid medium.

Total RNA was isolated from the cultured HUVECs from two donors A (Batch_A) and B (Batch_B) at population doubling levels 12 (PDL 12, young) and 39 (PDL 39, senescent) using TRIZOL reagent method (Invitrogen), respectively. Library was made with Illumina Truseq RNA Sample Preparation v2 Kit, then sequencing was carried out with Illumina HiSeq 2000 platform at BIOPIC at Peking University.
Sequencing reads that contained adapters or had low quality were pre‐filtered before mapping. Filtered reads were mapped to the hg19 genome using Tophat2 software (Version 2.0.11; Kim et al., 2013 (link)). Relative expression levels (reads per kilobase per million mapped reads, RPKM) were calculated by Cufflinks software (Version 2.2.1; Trapnell et al., 2010 (link)). Genes with RPKM <2 at both PDL12 and 39 were excluded in further analyses. The gene is defined as “transcriptionally changed or shifted” as its transcript is changed twofold in both Batch_A and Batch_B.
The senescence‐altered genes were subjected to enrichment analyses at Gene Ontology (GO; http://david.abcc.ncifcrf.gov/; Huang da et al., 2009 (link)).

Total RNA was extracted using TRIzol reagent method (Invitrogen). Complementary DNA (cDNA) was obtained via reverse transcription of equal amount of RNA following protocols and subsequently mixed with primers, SYBR Green PCR MIX (Applied Biosystems) and germ-free water. The reaction products were run on ABI Prism 7900HT sequence detection system (Applied Biosystems), and serial cDNA dilutions were used to generate GAPDH intensity reference standards according to the comparative Ct method11 (link). The primer sequences of all genes were listed in Table S5.