GSM8768319: PolyA_KO3; Mus musculus; RNA-Seq
The mice were euthanized through carbon dioxide induced asphyxiation lung samples were dissected and snap-frozen in liquid nitrogen. RNA was extracted from the tissue using TRIzol (ThermoFisher Scientific, catalog no. 15596018) and RNeasy Mini Kit (Qiagen, catalog no. 74104), according to manufacturers' protocols. Except where noted, we constructed all libraries according to the manufacturer's protocols and used 11 cycles of PCR during the final library amplification step. To allow us to assess duplication rates with UMI sequences, we used the xGen UDI-UMI adapters (IDT, catalog no. 10005903) for all libraries, diluting the initial 15 μM stock of adapters to 1.5 μM in NGS adapter buffer (10 mM Tris pH 8.0, 0.1 mM EDTA, 100 mM NaCl). We used the TruSeq Stranded mRNA library preparation kit (Illumina, catalog no. 20020594) and the TruSeq Stranded Total RNA Human/Mouse/Rat library preparation kit (Illumina, catalog no. 20020596) to prepare the PolyA and rRNA pull-down libraries, respectively. For the rRNA digestion libraries, we used the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit (Illumina, catalog no. 20040525). This kit uses a different strategy than the TruSeq kits for ligating indexed adapters to cDNA fragments that is not compatible with the xGen adapters. In order to use the same adapters, and their included UMIs, across all libraries, we modified the protocol for the Illumina Stranded Total RNA Prep Ligation as follows: 1) During the 'Ligate Adapters' step, replace the RNA Index Anchors with the diluted xGen adapters, and 2) during the 'Amplify Library' step, replace the Index Adapters with the PCR Primer Cocktail (PPC) from the TruSeq Stranded Total RNA kit. We performed all other steps according to the manufacturer's protocol. All PolyA and ribo-depleted libraries used 200 ng of input RNA. For the 'No Select' libraries, we used 15 ng of total RNA as input into the TruSeq Stranded Total RNA library preparation protocol, skipping the 'Deplete rRNA' step and starting with the 'Fragment and Denature RNA' step (see supplementary methods for detailed protocol). All remaining steps in the library preparation used reagents from the TruSeq Stranded Total RNA library preparation kit and followed that protocol. Since the majority of reads from the No Select library come from rRNA reads, we prepared and sequenced three No Select libraries from each sample/condition in order to get a sufficiently diverse sampling of non rRNA molecules.