Gel doc 2000 gel imaging system

Cells were harvested in RIPA lysis buffer (Thermo, MA, USA) containing protease and phosphatase inhibitors at 4°C. Protein extract was prepared according to the instruction of extraction kit (Beyotime Biotechnology Company, Shanghai, China). BCA protein assay kit (Beyotime Biotechnology Company, Shanghai, China) was used to quantify the protein. Then equal total amounts of proteins were separated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and transferred onto the PVDF membrane (Bio-Rad Company, USA). After blocking with 5% non-fat milk, the membranes were incubated with specific primary antibodies against the indicated protein overnight at 4°C, and washed with PBST, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were developed using the enhanced chemiluminescence reagent and scanned by the Bio-Rad Gel Doc 2000 gel imaging system with β-actin band as the reference.

Following RNA extraction, genomic DNA contaminants were removed via treatment with DNAse 1 for 45 min at 37 °C. Purified RNA was then reverse transcribed to cDNA using SuperScript III MMLV reverse transcriptase (Invitrogen) with random hexamers and oligo-dT primers. The cDNA was diluted appropriately and used as template for semi-quantitative PCR using Phusion polymerase according to manufacturer’s protocol using primers specific for AS events (see Table 1). PCR products were then run on an agarose (3 %) gel and visualized using Gel Doc 2000 Gel Imaging System (BioRad).Table 1

Murine RT-PCR primers used for AS event validation in this study

Gene Name	Primer Sequences (5’- 3)’
Hnrnpd	FW: GAAAGTATCCAGGCGAGGTG
	RV: GCTATTAGCAGGTGGCAGGA
Hnrpdl	FW: CAGACTACAGCGGTCAGCAG
	RV: TGGACCAATACCCCCTACAA
Srsf7	FW: CGCCTTGATTCAGAATGTCA
	RV: TGATCTTGACCTCCGTCCTC
Ogt (partial intron 4 retention)	FW: ACTGTGTTCGCAGTGACCTG
	RV: CAAATCTCCCCTTGTGCATT
Ogt (full intron 4 retention)	FW: CTTGGTAGCAGCAGGTGACA
	RV: AATGCTCACGGTCTTGCTTT
Slc35b1	FW: AAGGACCCAAACAGGAGACA
	RV: ATGGCACCCACATAGGAGAC
Ufd1l	FW: TGTTCATTTTATTTCAAAAATCGGAGC
	RV: AAGAACTCATCATAGTGCTCCTGC
Ivnsabp1	FW: AGCATCTGGGAGAATGGAGA
	RV: CATCATCACTGCCAAACACC
Casp4	FW: TGCTGAACGCAGTGACAAGC
	RV: TAAGAGCCTTTCGTGTACGGC
Nnt	FW: AACAGTGCAAGGAGGTGGAC
	RV: GTGCCAAGGTAAGCCACAAT
Nt5c3	FW: GCTGGCCCAGTACATATTCA
	RV: GGGCATCTTTTCCCATTGTA
Frrs1	FW: TTGCATTTCTCACGACCAG
	RV: TAGCCTCAGGAAGGGTGATG

The last batch of samples was used to analyse the microbial community using the method of polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Total genomic DNA was extracted according to the protocol [34 (link),35 (link)]. Bacterial 200 bp fragments of the 16S rRNA gene (V3 region) were amplified with the primers (338f, forward: 5′-ACTCCTACGGGAGGCAGCAG-3′; 534r, reversed: 5′-ATTACCGCGGCTGCTGG-3′). PCR was performed using a 25 µl (total volume) mixtures, containing 1 µl of each primer (12.5 pmol), 5 µl of each deoxyribonucleoside triphosphate (200 µmol), 5 µl of 10× PCR buffer (100 mmol Tris–HCl, 15 mmol MgCl₂, 500 mmol KCl, pH 8.3) and 0.5 µl of Taq DNA polymerase (Promega Co., USA), and made up to 25 µl with sterile water. PCR products were confirmed by 2.0% (w/v) agarose gel with ethidium bromide (0.5 µg l⁻¹) staining. Amplified DNA was then purified using a Qiaquick PCR cleanup kit (Qiagen Inc., USA). The PCR-amplified fragments were separated by DGGE using a Dcode universal mutation detection system (BioRad, USA). After electrophoresis, the gel was stained with ethidium bromide for 30 min and photographed with a Gel Doc 2000 gel imaging system (BioRad, USA).