Polya spin mrna isolation kit

Starting RNA was either “mouse liver control RNA” (Clontech), or was prepared from murine lymphocytes that were isolated from a B6CBF1 mouse spleen by homogenization through a cell strainer in DMEM-10 media, followed by centrifugation through Lympholyte Ficoll (Cedarlane). For single cell preparations, lymphocytes were additionally stained with anti-CD5 FITC antibody and sorted using a FACS aria fusion (BD Bioscience) into lysis buffer immediately before first-strand synthesis (see below). Otherwise, total RNA was extracted from the lymphocyte suspension using 1 mL Trizol (Ambion) and isolated with 500 μL chloroform before being ethanol precipitated. This was followed by poly-A purification for a selection of samples (Table S1) using the polyA Spin mRNA isolation kit (NEB) following the manufacturer’s instructions. ERCC spike-in probes were added to a subset of samples in the following way. 0.2 μL of a 1:10 dilution of ERCC spike-ins (Ambion) were added to 1 μL of 1μg/μL total RNA, before dilution to 100 pg/μL for low input samples, while 1 μl of a 1:10 dilution were included with the samples containing 100 ng of poly-A+ RNA (Table S1). The RNA was then divided into two equal samples for the different first-strand incubation temperatures.

Total RNAs from WT and NIPBL-MS LCLs were isolated with TRIzol Reagent. First the RNAs with polyA tails were separated from the rest of the RNA using the polyA spin mRNA isolation kit (NEB, S1560S), following the suggested protocol. The resulting RNA that mainly consisted of rRNA was divided into two portions. One portion of this RNA sample was subjected to ribo-depletion using the Ribo-Zero rRNA Removal kit (Epicenter, MRZH116), resulting in a pool of RNAs enriched for ncRNAs such as tRNAs, microRNAs, and snoRNAs.

PolyA RNA was isolated using the PolyA Spin mRNA Isolation Kit (New England Biolabs). RNA samples were purified as described above for five biological replicates of HUVEC. RNA was fractionated according to manufacturer’s instructions. cDNA was made from equal proportions of RNA based on the total mass collected from each fraction.

RNA of 10⁶ BMDMs was isolated using TRIzol reagent (Invitrogen) and resuspended in RNase-free water. Polyadenylated and non-polyadenylated RNA was enriched with polyA Spin mRNA isolation kit (NEB, S1560S) based on manufacturer’s protocol. RT-PCR was performed with same input volume, independent of concentration and normalized to non-polyadenylated RNA fraction.

RNA of 10⁶ ECs was isolated using TRIzol reagent (Invitrogen) and resuspended in RNase-free water. Polyadenylated and nonpolyadenylated RNA were enriched with the polyA Spin mRNA isolation kit (New England Biolabs, S1560S) based on the manufacturer’s protocol. Real-time PCR (RT-qPCR) was performed with same input volume, independent of concentration, and normalized to nonpolyadenylated RNA fraction.