Superscript 3

Total RNA was extracted using TRIzol (Life Technologies) and digested with RQ1 DNAse (M6101 Promega) following manufacturer’s instructions. For reverse transcription, 0.5 µg of RNA was heated for 5 min (min) at 65 °C and cDNA generated using SuperScript III (Life Technologies) and random primers for 1 h (h) at 50 °C, followed by heat inactivation for 15 min at 70 °C. qPCRs (for primers, see Supplemental Table S1) were performed with SYBR Green master mix (Ozyme) using a LightCycler 480 (Roche) and incubated for 2 min at 95 °C with 35 cycles of 10 s (sec) at 95 °C, 25 s at 60 °C, and 25 s at 72 °C. Data were normalized to GAPDH and analyzed using the 2^−ΔΔCT method^{40 (link)}. Poly(A) tail length was analyzed using the ePAT method^{41 (link)}. Briefly, 1 µg of total RNA was incubated with 5 μM oligo-(dT)-anchor (5′GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT3′) and 5 U of Klenow polymerase (New England Biolabs) for 1 h at 37 °C for template extension of the poly(A) tail, followed by reverse transcription using 200 U of SuperScript III (Life Technologies) for 1 h at 55 °C. Poly(A) tails were amplified by PCR using a gene-specific forward primer and the oligo-(dT)-anchor and migration of PCR product performed on 3% UltraPure Agarose 1000 gel (Life Technologies).

For measuring protein-coding mRNAs, reverse transcription (RT) was performed using SuperScript III according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA) followed by quantitative real-time PCR (RT-qPCR). For all genes, oligo-dT primer reverse transcription was performed using 350 ng of total RNA isolated from the human chondrocytes in a 20 μL RT reaction with SuperScript III, followed by qRT-PCR using 5 μL of 8-fold diluted RT reaction in 20 μL of qRT-PCR (ViiA 7 Real-Time PCR System, Thermo Fisher Scientific, Waltham, MA, USA). Transcript levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the results are presented as the relative abundance using the 2–ΔΔCT method (26 (link)). The primer sequences are listed in Supplementary Table 1.

SuperScript III (Thermo Fisher Scientific) was used to reverse transcribe 2 μg of RNA into cDNA using the random primers supplied with the SuperScript III kit. Fresh cDNA was diluted 1:10 with ddH₂O and was used immediately. Primers were designed by using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) and were selected to be located within the first half of the target gene, and to produce a product between 50 and 150 nucleotides in length. Real-time qPCR was performed with Power SYBR Green (Thermo Fisher Scientific). The Applied Biosystems ViiA 7 instrument was used with the following parameters for qPCR: (i) holding stage, 50°C for 2 min and 95°C for 10 min; (ii) PCR stage, 95°C for 15 sec and 60°C for 1 min, repeated 40 times with fluorescence recorded at 60°C; and (iii) melting curve stage, 90°C for 15 sec, 60°C for 1 min, then 95°C for 15 sec with the fluorescence recorded every 0.05 sec. Each reaction produced only one melting curve, indicating only one target was amplified during the qPCR. The threshold cycle (ΔΔC_T) method was used to determine the relative amount of RNA present in the sample tested. The atpI gene was used as an internal reference to normalize the CsrA-RNA coimmunoprecipitation because we have previously shown that CsrA does not bind to the mRNA transcript of this gene (17 (link)).

Using the pre-permeabilization mix for most tissue types from the protocol, the pre-permeabilization was carried out using 0.4% Collagenase 1 (Thermo Scientific, 17018-029) in BSA (Bionordika, B9000S) and HBSS buffer (Thermo Fisher Scientific, 14025-050) and was incubated for 20 min in 37 °C. The pre-permeabilization was followed by incubation in 0.1% pepsin-HCl (Sigma-Aldrich, P7000-25G, pH 1) for 10 min at 37 °C to permeabilize the tissue. A cDNA-mix containing Superscript III (Thermo Fisher, 18080085), RNaseOUT (Thermo Fisher, 10777019), 0.1 M DTT (Thermo Fisher, included with Superscript III), dNTPs (Thermo Fisher, R0191), BSA (Bionordika, B9000S), and Actinomycin D (Sigma-Aldrich, A1410-2MG) was added and the slide incubated at 42 °C overnight (~18 h). The tissue was washed with 0.1× SSC between each incubation step.

Primers were designed to amplify at least three consecutive exons of MANE (matched annotation between NCBI and EBI) transcripts, with an RT-PCR product size between 340 to 500 bp, except for BAG3, KCNH2 and TTN, which amplified larger products due to large exon sizes. One microgram of RNA was reverse transcribed using 200 U Superscript™ III (Invitrogen, Massachusetts, United States), 4 µl Superscript III™ 5X first-strand buffer, 5 mM DTT, 40 U RNaseOUT™ (Thermo-Fisher), 0.25 mM dNTP (Roche, Basel, Switzerland) and 0.01 mM random hexamer primer (Thermo-Fisher) in a final volume of 20 µL. Complementary DNA products were diluted 1:2 in DEPC-treated water and amplified as previously described^{5 (link)}, using an annealing temperature of 60 °C unless otherwise specified (Supplementary Table 5). PCR products were resolved on an agarose gel and all gel images were derived from the same experiment. PCR products were purified using 5 U Antarctic Phosphatase (New England BioLabs, Massachusetts, United States) and 20 U Exonuclease I (New England BioLabs), and Sanger sequenced at the Macrogen Sequencing Facility (Seoul, Republic of Korea). Chromatograms were analysed using Sequencher™ v.5.4.6 (Gene Codes Corporation, Michigan, United States).