alias: GSM1869998
title: GSM1869998: Mac.4.3; Homo sapiens; miRNA-Seq
library_construction_protocol: At 1, 4 and 24 hours post-infection, infected macrophages were washed twice with PBS and homogenized by adding Trizol reagent (Sigma). All the aliquots were stored at -80°C until required for RNA extraction. RNA extraction was performed by adding 200uL of chloroform (Sigma) to each sample and mixing by vortexing for 60 sec. The solution was then centrifuged for 15 min at 12000xg at 4°C. The aqueous phase was removed, transferred to a separate tube containing 500uL of isopropanol (Sigma), gently mixed and centrifuged 10 min at 12000xg at 4°C. The supernatant was discarded, the RNA pellet was resuspended in 1mL of 75% Ethanol, mixed by vortexing and centrifuged 5 min at 7500xg at 4°C. After discarding all the supernatants, the RNA was transferred to a miRNeasy mini kit column (Qiagen, Ldt.) and further purification steps were conducted according to the manufacturer’s instructions. In addition, the RNA was subjected to an on-column DNase treatment step (Qiagen Ltd.) to remove any DNA residues that could affect the downstream reaction. RNA quantity and quality was assessed using a NanoDrop TM 1000 spectrophotometer (Thermo Fisher Scientific Inc.) and on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (Agilent Technologies, Ltd.). All samples displayed a 260/280 ratio greater than 2.0 and RNA integrity numbers (RIN) greater than 7,5. Small RNA-seq libraries were prepared using the Illumina TruSeq Small RNA Sample Preparation Kit according to the manufacturer’s protocol using 1ug of total RNA (extracted from not-infected and infected samples not bead-beaten). Briefly, RNA adapters were ligated to the 5’ and 3’ ends of the small RNA molecules, followed by reverse transcription and 11 cycles of PCR amplification by using indexed primers. The libraries were size-selected on a 6% polyacrylamide gel for the desired size of 147-157 bp and then purified from the gel. The molarity and size of finished miRNA-seq libraries were quantified on an Agilent 2100 Bioanalyzer using an Agilent High Sensitivity DNA chip (Agilent technologies). Libraires were loaded at a concentration of 10pM onto the flowcell and were single-end sequenced on Illumina HiSeq 2000 or Illumina MiSeq.
identifiers.namespace: GEO
identifiers.id: GSM1869998