GSM2870831: 53M1 [miRNA-seq]; Mus musculus; miRNA-Seq
For RNA extraction, ¾ of the extracted sperm was incubated at 60 °C for 15 mins with slow rotation in 33.3 µl sperm lysis buffer (6.4 M Guanidine HCl, 5% Tween 20, 5% Triton, 120 mM EDTA, 120 mM Tris pH 8.0) with 3.3 µl Proteinase K (19 mg/ml) and 3.3 µl 0.1 M DTT. After the incubation, one volume (100 µl) of ultra-pure water was added. 700 µl Qiazol Lysis reagent (QIAGEN, Cat. 79306) was added and samples were vortexed for 5 mins. 140 µl chloroform was added and samples were shaken vigorously for 30 seconds before 3 mins incubation at RT. Samples were centrifuged at 12,000 x g at 4 °C for 15 mins then the upper aqueous phase was transferred to a new reaction tube. One volume of 70% EtOH was added and mixed thoroughly. Samples were transferred to a RNeasy mini spin column and the protocol from the miRNeasy Mini Kit (Qiagen, Cat. 217004) was then followed, including the separation of the small RNA and large RNA fractions using an RNA MinElute spin column (Qiagen, Cat. 74204). The small RNA fraction was eluted in 14 µl RNase-free water and quantified using the microRNA kit from Qubit® (Cat. Q32880). 6 µl of the small RNA fraction was used for small RNA library preparation using the NEBNext® Small RNA library prep set for Illumina (Cat. E7330S) as per the protocol. Each sample was uniquely barcoded using one of the NEBNext® Index Primers for Illumina (Cat. E7300S, E7580S, E7710S, E7730S). For PCR amplification, 15 cycles were used. Libraries were purified using the QIAQuick PCR purification kit (Qiagen, Cat. 28104) and DNA was eluted into 32 µl nuclease-free water. An aliquot of each library was diluted and 1 µl was run on an Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA kit (Cat. 5067-4626) to assess the size distribution of the library. The libraries were pooled using equal volumes and size selected for between 140-200 bp (corresponding to an insert size of 13-73 bp) using a BluePippin machine (Sage Science) with 3% agarose cassettes (Sage Science, Cat. BDF3010).
GEO Accession
GSM2870831