S1: Preparation of knock down cells

We have knocked down TRF1, TRF2, PARP-1 and SLX4 transiently by transfection as per manufacturer’s protocol. For transfection, 0.2 million cells were counted and seeded in 35mm petridish. After overnight incubation, medium was replaced with a reduced serum medium, Opti-MEM (Invitrogen, life technologies). The cells were transfected with the Silencer Select siRNAs (Ambion, life technologies) specific for a target gene and with a negative control (scrambled version) using lipofectamineRNAiMAX Reagent (Invitrogen, life technologies) according to manufacturer’s protocol. All the experiments were performed 48 hrs post transfection.

List of siRNA used for Gene knockdown

Sl no / Gene / Product code / SiRNA (sense sequence 5’-3’)
1 / TRF1 / s13988
s13989 / siRNA1=>ACCUAGUGGUAAUGAUGUUdTdT,
siRNA2=>GCAGAAUACCUGUUUCAAAdTdT
2 / TRF2 / s13991
s13992 / siRNA1=>GGUCGAAUCCAGUAGAAAAdTdT,
siRNA2=>GUACAACCAAUAUAACAAAdTdT
3 / PARP-1 / s1097
s1098 / siRNA1=>GGUGAUCGGUAGCAACAAAdTdT
siRNA2=>AGAGCGATGCCTATTACTGdTdT
4 / SLX4 / s39052
s39053 / siRNA1=>CAUCCAAACUGAACGAAGAdTdT
siRNA2=>CCAAAGGUGCUAAUCGGAAdTdT

S2: RNA extraction and Q RT-PCR

Total RNA was isolated from transfected cells using TRIzol Reagent (Invitrogen; life technologies) following standard protocol. The RNA was treated with RNase free DNase (Sigma) for 1hr at 37°C to avoid the DNA contamination. The cDNA was prepared from 1µg of RNA from all samples using M-MLV reverse transcriptase (NEB) and random hexamer (NEB). The quantitative PCR analysis was done on StepOnePlus™ Real-Time PCR System (Applied Biosystem; Life Technologies). Expression of telomere-associated genes and TERRA were done using Taqman probe and SYBR green respectively.

Gene expression of TRF1, TRF2, TIN2, POT1, PARP-1, SLX4 was done by Taqman assay (Thermo Fischer Scientific) and the reactions were performed under standard assay programme (95 °C for 10 min and then 40 cycles of 95 °C for 15 s, followed by annealing and extension at 60 °C for 1 min). The internal control was TERRA expression from 10q, 15q, XpYp and XqYq was measured using SYBR green. The threshold fluorescence signal was set up manually and the corresponding Ct values were determined. The expression levels were normalized using 18S rRNA (for telomere-associated proteins) and 36B4u (for TERRA) respectively as endogenous control by ΔΔCt method.

List of Taqman Assays used in q-RT PCR

Taqman Assay ID / Gene Symbol
Hs00242302_m1 / PARP1
Hs00819517_mH / TERF1
Hs00194619_m1 / TERF2
Hs00536164_m1 / SLX4
Hs99999901_s1 / 18S
Hs01554309_g1 / TINF2
Hs00209984_m1 / POT1

List of Primers used for SYBR-green q-RT PCR

TERRA / Forward 5’-3’ / Reverse 5’-3’
TERRA-10q / GAATCCTGCGCACCGAGAT / CTGCACTTGAACCCTGCAATAC
TERRA 15q / CAGCGAGATTCTCCCAAGCTAAG / AACCCTAACCACATGAGCAACG
TERRAXpYp / GCAAAGAGTGAAAGAACGAAGCTT / CCCTCTGAAAGTGGACCAATCA
TERRAXqYq / GGAAAGCAAAAGCCCCTCTGAATG / ACCCTCACCCTCACCCTAAGC
36B4u / CAGCAAGTGGGAAGGTGTAATCC / CCCATTCTATCATCAACGGGTACAA

S3: Depletion of target proteins by siRNA

We confirmed depletion of three proteins like PARP-1, SLX4 and TRF2 by respective siRNAs using western blot. A typical picture is shown in Figure S3. PARP-1, SLX4 and TRF2 was depleted about 91%, 72% and 88% respectively after transfection of respective siRNAs. We did not get any difference of expression of each protein between untreated cells and cells transfected with scrambled version siRNA – the negative control of this experiment. Beta actin (B-actin) was used as the internal control of this experiment.

Figure S3