GSM9438416: G2m4_4; Mus musculus; RNA-Seq
Lung accessory lobes were collected in RNAprotect tissue reagent (Qiagen) overnight before removal from the reagent and storage at -80oC until RNA extraction. RNA was extracted from the acessory lobe by homogenising the tissue in RLT buffer (+1% BME) using a Precellys 24 Tissue Homogenizer. The homogenate was centrifuged at full speed and RNA was extracted from the supernatant using the RNeasy Mini kit (Qiagen), including an on-column RNase-free DNase digestion step (Qiagen). Total RNA quantification and integrity was confirmed using Agilent's 4200 Tapestation (Standard Total RNA assay). For each sample, 500ng of total RNA were processed using the KAPA mRNA HyperPrep Kit (Roche p/n KK8580) according to manufacturer's instructions. Briefly, mRNA was isolated from 500ng total RNA by use of paramagnetic Oligo dT beads to pull down poly-adenylated transcripts. The purified mRNA was fragmented using chemical hydrolysis (heat and divalent metal cation) and primed with random hexamers. Strand-specific first-strand cDNA was generated using reverse transcriptase in the presence of actinomycin D. The second cDNA strand was synthesised using dUTP in place of dTTP, to mark the second strand. The resultant cDNA was then “A-tailed” at the 3' end to prevent self-ligation and adapter dimerisation. Full-length xGen adaptors (IDT), containing two unique 8-bp sample specific indexes, a unique molecular identifier (N8) and a T overhang are ligated to the A-tailed cDNA. Successfully ligated cDNA molecules were then enriched with limited cycle PCR (11 PCR cycles). The high fidelity polymerase employed in the PCR is unable to extend through uracil. This means only the first strand cDNA is amplified for sequencing, making the library strand specific (first-strand). High yield, adaptor-dimer free libraries were confirmed on the Agilent TapeStation 4200 (High Sensitivity Agilent DNA 1000 assay) and a library quantification was estimated using the Qubit dsDNA HS assay (Life Technolgies). Libraries were normalised to 4nM and then an equal volume of each were pooled together. The library pool was denatured and sequenced on the NextSeq 2000 instrument (Illumina, San Diego, US) at 750pM, using a 56bp paired-read run with corresponding 8bp unique dual sample indexes and 8bp unique molecular index.