Agilent 2200

Two colon tissues were collected from each group. Total RNA was extracted from the colon tissues using the TRIzol method. The purity and concentration were detected using the NanoDrop 2000. The RNA integrity was detected using the Agilent 2200. Total RNA samples with RIN (RNA integrity number) values higher than 8 were used for library construction. A total of 3 μg of total RNA from each sample was used to construct the small RNA library. Different index tags were selected for library construction based on the operational manual of the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA). The quality of the library after construction was examined using the Agilent 2200.

Total RNA was extracted from each sample using an RNAprep pure Plant Kit (Tiangen, Beijing, China), according to the manufacturer’s protocol. The RNA concentration of each sample was measured using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). The RNA quality was assessed using an Agilent2200 (Agilent Technologies, Santa Clara, CA, USA).
The sequencing library for each RNA sample was prepared using a TruseqTM RNA sample prep Kit (Illumina, San Diego, CA, USA), following the manufacturer’s protocol. Briefly, mRNA was purified using poly-T oligo-attached magnetic beads (Invitrogen,Carlsbad, CA, USA) from 5 μg total RNA. The mRNA was fragmented, and the RNA fragments were reverse transcribed and amplified to double-stranded cDNA. Index adapters were then ligated to the cDNA according to the protocol of the TruseqTM RNA sample prep Kit (Illumina). The library was quantified using a TBS-380 mini-fluorometer (Picogreen, Cohasset, MA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System, using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and a sequence length of 2*101 bp paired-end reads were generated.

Total RNA from DANCR knockdown and control HuCCT1 cells were isolated and quantified. The concentration of each sample was measured with NanoDrop 2000 (Thermo Scientific, USA). Quality evaluation was conducted with Agilent2200 (Agilent, USA). The sequencing library of each RNA samples was prepared using Ion Proton Total RNA-Seq Kit v2 (Life technologies, USA). See Supplementary Tables S2 and S3 for the data of the six samples.

Trizol (Invitrogen) was used to extract RNA from cells, after which an Agilent 2200 machine was used to assess RNA quality prior to storage at −80°C. RNA samples with an integrity score >7.0 were used to prepare cDNA libraries.
To produce small RNA sequencing libraries, we utilized an NEBNext Small RNA Library Prep Kit for Illumina. Briefly, RNA was ligated to the provided 5′ and 3′ adapters, followed by first strand cDNA synthesis. Index PCR was then used in order to apply index sequences and Illumina sequence adapters. Library purification was then performed, and a Bioanalyzer 2200 (Agilent, CA, USA) was used for quality control followed by sequencing on a HiSeq X‐ten platform (Illumina, CA, USA) using 150 bp paired‐end reads.

ChIP-seq libraries were generated for pair-end sequencing using the TruSeq DNA LT
Sample Prep Kit (Illumina, San Diego, CA), according to the manufacturer’s
instructions. Briefly, the fragmented DNA samples (1 μg/each, in duplicate) were
end-repaired, A-tailed at the 3’end and ligated with indexed adapters provided.
The potential target DNA samples were extracted using AMPure XP magnetic beads
and amplified by PCR to create the final ChIP-seq libraries, which were
quantified by Agilent 2200. The DNA in the ChIP-seq libraries was sequenced
twice in the Solexa sequencer (PE150), according to the manufacturer’s
instructions (Illumina).

Total RNA was extracted from each sample using TRIzol Reagent (Life technologies) according to the protocol from manufacturer. The concentration of each sample was measured by NanoDrop 2000 (Thermo Scientific). The quality was assessed by the Agilent2200 (Agilent) (S3 Table). The RNA integrity was assessed by electrophoresis with denaturing agarose gel.

Total RNA was extracted from cells with Trizol (Invitrogen) according to the manufacturer’s protocol and purified by mRNA enrichment. mRNA poly(A) tails then enriched with oligodT magnetic bead. rRNAs were removed. rRNA hybridized with DNA probe. RNaseH chose to digest DNA/RNA hybridization chain. DNase-digested DNA probe resulted in purified RNA. The cDNA libraries were constructed for each pooled sample with VAHTSTM Total RNA-seq (H/M/R) according to the manufacturer’s instructions. Final cDNA libraries were created by PCR purification and enrichment, then quantified by Agilent2200. The tagged cDNA libraries were pooled in equal ratio and used for 150 bp paired-end sequencing in a single lane of Illumina HiSeq Xten by NovelBio Corp. Laboratory. NovelBrain Cloud Analysis platform was used to analyse high-throughput sequencing data. All RNA-Seq reads were mapped to mouse genome-using TopHat2. Transcript abundance was quantified using Cufflinks and annotations from Ensembl release 70, and FPKM (fragments/kb of transcript/million fragments mapped) values were calculated. To minimize dispersion effect by low-FPKM values, all the FPKM values were modified by addition of 0.1 in log2 transformation. Gene ontology analysis for biological process of the selected genes was performed using Partek Genomic Suite (Ryoka systems).