title: GSM1155646: 1199_NM; Mus musculus; RNA-Seq
library_construction_protocol: Approximately 200 mg tissue sample was placed in 1.5 ml RNAlater buffer (Ambion) and snap-frozen in liquid nitrogen. At time of extraction, samples were centrifuged for 10 min at 16.000g at room temperature. Pellets were resuspended in 150 μl of Lysis Buffer (Tris/HCl pH 8, 1 mM EDTA, 15 mg/mL Lysozyme (Sigma), 15 μl Proteinase K (20 mg/mL)) and incubated at room temperature for 10 min with brief mixing every 2 min. Following addition of 1.2 mL QIAGEN RLT buffer containing 1% v/v β-mercaptoethanol, 1 mL of 0.1 mm glass beads (BioSpec) were added and samples were homogenized in a FastPrep at setting 5 (4 pulses of 20 sec). Samples were kept on ice for 1 min between pulses. Lysates were then homogenized with a QIAshredder spin columns and RNA was isolated using the AllPrep mini kit (Qiagen), according to the manufacturers protocols, which included an on-column digestion with DNase I. 25 μg of RNA from each tissue sample was processed with the MICROBEnrich kit (Ambion) and 5 μg of processed RNA was further depleted of rRNAs using the Meta-Bacteria RiboZero rRNA removal kit (Epicentre). rRNA depleted RNA was prepared for Illumina paired-end sequencing using the NEB Next mRNA Library Prep Master Mix Set for Illumina (NEB, E6110S); manufacturer’s protocols were followed with the following modifications. RNA was fragmented for 10 minutes, and then purified with Qiagen RNeasy MinElute spin columns. Reverse transcription was performed with SuperScript III (Invitrogen). Library preparation reactions were cleaned up using Agencourt AMPure XP beads. Size selection was performed before ligation mediated PCR using Invitrogen E-Gel 2% with SYBR Safe staining. Excised gel fragments were purified with Qiagen QIAQuick Gel Extraction kit. Adapters and primers were synthesized by IDT following published Illumina sequences. Enrichment PCR was performed with Kapa HiFi HotStart ReadMix. Primers were used at a final concentration of 500 nM; cycling parameters were as follows: 94 oC for 5 min, 15 cycles of 94 oC 1 min, 62 oC for 30 sec, 72 for 45 sec and then 72 oC for 10 min. Libraries were quantified using the Agilent BioAnalyzer DNA 1000 chips, diluted to 12 pM and sequenced for 100 cycles (paired-end) on the Illumina HiSeq 2000 using standard methods.
attributes.tag: GEO Accession
attributes.value: GSM1155646