P-GSE43667-3 - P-GSE43667-3
nucleic acid library construction protocol
Total RNA was isolated from endometrium using TRIzol® (Invitrogen, Carlsbad, CA, USA) according to the manufacturers recommendations. Purity (based on 260nm:280nm and 260nm:230nm ratios) and quantity of the obtained total RNA was measured by use of a NanoDrop ND-1000 (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Integrity of the RNA was assessed by analysis on Agilent RNA Nano 6000 microfluidic chips with an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). RNA integrity numbers (RIN) ranged from 7.0 to 8.8. Equal amounts of total RNA from samples derived from proximal, medial and distal endometrial sections of one uterine horn were pooled for each animal. The mRNA-Seq sample preparation kit (Illumina, San Diego, USA) was used for preparation of RNA-Seq libraries. Library preparation followed the manufacturers instructions. Briefly, poly(A)-containing RNA was purified with oligo-dT-coated magnetic beads starting from 5 µg total RNA and fragmented under elevated temperature using divalent cations. The obtained cleaved RNA fragments were reverse transcribed to first strand cDNA using Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) and random primers, followed by second strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments underwent an end-repair process with T4 DNA polymerase, Klenow DNA polymerase and T4 Polynucleotide kinase, addition of a single ´A´-base, and ligation of adapters. Ligation products were subsequently separated on a 2% agarose gel (Biozym Phor Agarose, Biozym Scientific GmbH, Hess. Oldendorf, Germany) and a gel slice was cut out (X-Tracta, Biozym Scientific GmbH) in the 200 bp (±25 bp) range. After isolation of the DNA from the gel slice (QIAquick Gel Extraction Kit, Qiagen, Hilden, Germany), cDNA fragments were amplified through 15 cycles of PCR (Phusion DNA polymerase, New England Biolabs GmbH, Frankfurt a. Main, Germany) to generate the final sequencing libraries. Concentration of the cDNA fragments was estimated on an Agilent DNA 1000 chip (Agilent Technologies) and with the Qubit Fluorometer (Invitrogen).