Random primer

Bleomycin sulfate (Bleocip, Cipla), ketamine hydrochloride, xylocaine, anti-goat-IgG (SAB3700288, Sigma Life Science), TLR-2 (Sc-10739, Santa Cruz, USA), TLR-4 (Sc-16240, Santa Cruz), CD-68 (ab125212, Abcam), NF-κB-p65 (Sc-109, Santa Cruz), ExtrAvidin® Peroxidase (Extra-2,3, Sigma), NovaRED (SK-4800, Vector labs, USA), Meyer’s hematoxylin, TRIzol® (Invitrogen 15596018), chloroform, isopropanol, MMLV (M0253S, NEB), RNase (M0314, NEB), dNTPs (N0447S, NEB), random primers (S1330S, NEB), SYBR Green (S4438, Sigma), protease inhibitor (Sigma), hyaluronan quantikine ELISA (LMW-HA < 35–950 kDa, DHYAL0, R&D Systems, USA), and Lamin-A/C (612162, BD Biosciences, India) were used.

For cell lines, total RNA was isolated using Direct‐zol™ RNA MiniPrep kits (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions, and 0.5 μg RNA was reversely transcribed into complementary DNA (cDNA) by RevertAid™ Reverse Transcriptase (Fisher Scientific, Pittsburgh, PA, USA) with random primers (New England Biolabs, Ipswich, MA, USA) in a 10 μL reaction system. For serum samples, total RNA was isolated using TRIzol LS (Invitrogen, Carlsbad, CA) following the manufacturer's protocol, and reverse transcription was conducted using the Prime Script™ RT Reagent Kit (Takara, Dalian, Liaoning). The resultant cDNA then served as template for qRT‐PCRs. The expressions of LINC00310 and c‐Myc were specifically detected using the SYBR Green method with primers listed in Table S1. GAPDH or β‐actin was used as an internal control. Delta‐delta Ct values were used to calculate their relative expressions as previously described.28

Total RNA was isolated using the total RNA Miniprep kit (NEB, catalog no. T2010S) following the manufacturer's protocol. RNA (5 µg) was reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen, catalog no. 28025013) and random primers (NEB, catalog no. S1330S). cDNA concentrations were normalized and RT-qPCR was performed using specific primers and GoTaq qPCR master mix (Promega, catalog no. A6001) according to the manufacturer's specifications on the AriaMX Real-Time qPCR machine (Agilent). Expression of target mRNAs was normalized to β-actin and relative gene expression was calculated using ΔC_t and 2^−ΔΔC_t.

The construction of cDNA libraries and sequencing were performed essentially as previously described [34 (link)]. Briefly, a total amount of 1 μg RNA per sample was used as input material for RNA sequencing. The sequencing library was generated using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) by following the manufacturer’s recommendation. Briefly, mRNA was enriched from total RNA using Oligo (dT) magnetic beads, and fragmented in NEBNext First Strand Synthesis Reaction Buffer (5 ×) by ultrasound. First strand cDNA was synthesized with ProtoScript II Reverse Transcriptase using NEBNext Random Primers. Second-strand cDNAs were synthesized with the Second Strand Synthesis Enzyme Mix in the Second Strand Synthesis Reaction Buffer. Then double-stranded cDNA fragments were purified with 1.8X Agencourt AMPure XP Beads (NEB), end repaired, and ligated to Illumina sequencing adapters. The ligation products were purified with AMPure XP Beads, PCR amplified, and sequenced by Gene Denovo Biotechnology Co. (Guangzhou, China) using Illumina HiSeq 2500 to generate 150 bp paired-end reads.

The MCF7-LR cells were treated with the 76-E6 antibody or the control IgG for 24 h, and then RNA was extracted from the treated cells using the TRI reagent (Sigma-Aldrich). The reverse transcription was performed using 1.5 μg extracted total RNA per condition, random primers (New England Biolabs), and the SuperScript II reverse transcriptase (Thermo Fisher Scientific). The cDNA samples were analyzed with the SYBR Green Supermix (Quanta Biosciences) according to the manufacturer’s instructions using the Mx3005P thermocycler (Stratagene). The primers used in the study are listed in Table S3.

To test if dCas9-SAM could activate endogenous target genes when recruited to the sites where we added the GBSs, we created gRNAs containing MS2 loops by cloning the target sequence (Table 6) into the sgRNA(MS2) plasmid (#61424; Addgene). Next, U2OS-GR cells were transfected with 600 ng each of the MS2-containing gRNA, dCas9-VP64 expression construct (#48223; Addgene), and an MS2-p65-HSF1 activator expression construct (#61423; Addgene) by nucleofection (Lonza Nucleofector kit V) according to the manufacturer’s instructions. 24 h after transfection, total RNA was isolated using the RNeasy kit (QIAGEN), DNase-I digested and reverse transcribed using random primers (NEB) and analyzed by qPCR using primers listed in Table 7. To calculate the fold up-regulation, we first calculated the RNA level of the targeted gene relative to RPL19 (which served as an internal control) observed when cells were transfected with the promoter-targeting gRNA. In addition, we determined the RNA level of the targeted gene for each of three guide RNAs that target the promoter of another gene. Finally, the fold regulation by the targeting gRNA is determined by dividing the RNA level for the targeting gRNA by the average RNA level for the three non-targeting gRNAs.