Dnaase 1

RNA was extracted 48 h after transfection using Trizol (Invitrogen, Waltham, MA, USA, cat. number 15596018) as described in the manual (MAN0016385) and DNA digested with DNAase I (Invitrogen, Waltham, MA, USA, #18068015). The presence of the 28S and 18S was evaluated on 1% agarose gels. The RNA was quantified on a spectrophotometer, and a PCR was performed employing the GAPDH primers to ensure that no DNA was left. Then 1 μg of RNA per sample was used for retro-transcription using the RevertAid First Strand Kit (Thermo ScientificTM, Waltham, MA, USA, #K1691). cDNA was obtained and the qRT PCR was performed by using Thermo Maxima SYBR Green/ROX 1qPCR Master Mix (Thermo ScientificTM, Waltham, MA, USA, #K0251). The following primers were used: E6-18-F: 5′-GCG ACC CTA CAA GCT ACC TG-3′, E6-18-R: 5′-GTT GGA GTC GTT CCT GTC GT-3′; E7-18-F: 5′-AAC ATT TAC CAG CCC GAC GA-3′, E7-18-R: 5′-TCG TCT GCT GAG CTT TCT AC-3′; E6-16-F: 5′-ACT GCA ATG TTT CAG GAC CC-3′, E6-16-R: 5′-TCA GGA CAC AGT GGC TTT TG-3′; E7-16-F: 5′-CCC AGC TGT AAT CAT GCA TG-3′, E7-16-R: 5′-TGC CCA TTA ACA GGT CTT CC-3′; GAPDH-F: 5′-AAG GTC GGA GTC AAC GGA TTT-3′, GAPDH-R: 5′-CCA TGG GTG GAA TCA TAT TGG AA-3′. PCR cycles were as follows: 95 °C/10 min, 35 cycles: 95 °C/35 s; 60 °C/ 35 s; 72 °C/35 s. Data were processed with the Delta CT method.

RNA was extracted from tissue samples using TRIzol reagent (Invitrogen, China) as previously described [22 (link), 23 ]. Contaminated genomic DNA was removed by DNAase I treatment (Invitrogen, China). For qRT-PCR, RNA was reverse transcribed to cDNA by using a Reverse Transcription Kit (Takara, Dalian, China). Real-time PCR analyses were performed with Power SYBR Green (Takara, Dalian China) and the primers for each gene were designed as previously described [24 (link)]. Results were normalized to the expression of GAPDH. The relative difference in the expression level was calculated using the ^ΔΔCT method. The data presented are representative of three independent biological repeats each assayed in triplicate and are relative expression levels.

FACS-sorted cells were resuspended in RNA lysis buffer (Zymo Research, Irvine, CA) and stored at − 80 °C until RNA isolations were performed using the ZR RNA isolation kit (Zymo Research), according to the manufacturer’s instructions. The RNA was treated with DNAase I (amplification grade; Invitrogen) to remove any genomic DNA before being reverse-transcribed into cDNA, as described elsewhere [40 ]. To check for genomic DNA contamination control samples without reverse transcriptase were included. cDNA was stored at − 20 °C until further use. Real-time PCR was performed on a CFX96 system (Bio-Rad, Veenendaal, Netherlands) using SYBR Green reaction mix (Sigma-Aldrich) and Siglec expression was calculated relative to GAPDH expression. Primer sequences (Sigma-Aldrich) were derived from the Harvard Primer Bank database (Supplementary Table 1) [41 (link)].

For qRT-PCR total RNA was isolated with Trizol Reagent (Life Technologies, Gaithersburg, MD, USA) according to the manufacture’s guidelines. RNA was digested with DNAase I (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA using High Capacity RNA-to cDNA Kit (Applied Biosystems, Life Technologies, Gaithersburg, MD, USA). Quantitative RT-PCR was performed using SYBR green detection (Applied Biosystem, Life Technologies, Gaithersburg, MD, USA) and the ∆∆Ct method for relative quantification. Expression of β-actin was used as internal control.
The primers used for individual genes are listen in Additional file 1: Table S1.

RNA was extracted using the TRIzol method (Invitrogen) according to manufacturer's instructions and eluted with 0.1% diethylpyrocarbonate (DEPC)-treated water. Each sample was treated with DNAase I (Invitrogen). Total RNA concentration was quantitated by spectrophotometry; 1 μg of total RNA was used to reverse transcription using iScript^TM cDNA synthesis kit (Bio-Rad, Hercules, CA) according to manufacturer's instructions. For microRNA Taqman assays, 2.5 ng of total RNA were reverse transcribed using Taqman® MicroRNA Reverse Transcription Kit (Applied Biosystems).