Rna clean and concentration kit

RNA was extracted as described above and concentrated using an RNA Clean and Concentration kit (Zymo Research, Irvine, CA, USA). All samples were normalized with nuclease free water to a concentration of 50 ng/µL. 250 µg of RNA was synthesized into cDNA using Superscript III reverse transcriptase (Invitrogen|Thermo Fisher Scientific, Waltham, MA, USA) as previously described [19 (link)]. To identify any genomic DNA contamination, non-template controls of each RNA sample were also prepared and verified by reverse transcriptase PCR (RT-PCR) using GAPDH primers [42 (link)]. All contaminated samples were discarded. Quantitative reverse transcriptase PCR (qRT-PCR) was performed using Sybr green reagent (Applied Biosystems|Thermo Fisher Scientific, Waltham, MA, USA) using primers for CCL20, CERS2, CSF2, ICAM-1, IL-1α, IL-1β, IL-6, IL-8, MMP1, MMP9, TNFα [42 (link),43 (link),44 (link),45 (link),46 (link),47 (link),48 (link),49 (link),50 (link),51 (link),52 (link)]. All gene reactions were normalized to GAPDH [42 (link)], and analyzed using the ΔΔCT method. All experiments were performedat least three independent times.

We first treated total BAT RNA from n = 3 samples in the
IBA, LT, EAr, and SpW groups with DNase I (Invitrogen, Carlsbad, CA). A portion of
this RNA was saved for direct conversion into cDNA while the rest was fractionated
based on poly(A) tail length into short (≈25 nt) and long (>25 nt)
poly(A) tail fractions using the PolyATtract mRNA Isolation System IV (Promega,
Fitchburg, WI) as described (Meijer et al.,
2007). The resulting RNA fractions were concentrated using the RNA Clean
and Concentration Kit (Zymo Research, Irvine, CA). Random hexamer primed cDNA was
synthesized using SuperScript III (Invitrogen). 3′ RACE cDNA was
synthesized (Peddigari et al., 2013 (link)) with
the reverse primer
5′-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3′. ePAT and
TVN-PAT (TVN) cDNA (Janicke et al., 2012 (link))
from one poly(A) short and one poly(A) long RNA sample in each group was
synthesized using the ePAT primer
5′-GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT-3′; the TVN-PAT primer was the
same, except that it had a (VN) added to the 3′ end.

RNA extraction from whole cells was performed using QIAzol Lysis Reagent (QIAGEN) according to the manufacturer’s instructions. To eliminate DNA contaminations, RNA was treated twice with RNase-free DNase-I (Promega), and then purified with the RNA Clean and Concentration kit (ZYMO Research). After library preparation using Illumina TruSeq Stranded Total RNA with Ribo-Zero GOLD, the resulting cDNA was paired-end sequenced by IGA Technology Services [37 ] through an Illumina HiSeq2000 platform.
RNA-seq reads were mapped, with the same Razers3 parameters as the ChIP and input datasets, on a reference composed of a dimer of the 37cen consensus sequence (“SAT_EC” on repbase) and on 442 bp long portions of the following transcripts: TUBB (XM_001491178.5, nucleotides 488 to 929), PRKCI (XM_014732748.1, nucleotides 605 to 1046), TERC (NR_001566.1 nucleotides 9 to 450), TK (XM_001491081.5 nucleotides 26 to 467). The same length was used for each sequence in order to have comparable read counts without normalization.

Tissue samples were placed in 1 mL of Qiazol reagent (Qiagen, Hilden, Germany) and mechanically homogenized using a Tissue Ruptor (Qiagen). Total RNA preparation was then carried out following the manufacturer’s instructions. RNA samples were treated twice with 1 U DNase I (Promega, Madison, WI, USA) per μg RNA at 37 °C for 30 min. Following each DNase treatment after, the RNA was purified with the RNA Clean and Concentration kit (Zymo Research, Irvine, CA, USA) according to manufacturer’s instruction.