title: GSM3672453: CD87-_group2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from sorted cells using the AllPrep DNA/RNA Mini kit (Qiagen, Valencia, CA, USA) and subjected to Smart-seq2 protocol. In brief, the mixture of 1 µl of 10 mM oligo-dT30VN primer (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3'), 1µl of 10mM dNTP mix and 10ng extracted RNA in 5ul reaction volume was incubated at 72˚C for 3 min. Then, 5 μl of the reverse transcription mixture (200U SuperScript II reverse transcriptase, 20U RNase inhibitor, 1x Superscript II first-strand buffer, 5 mM DTT, 1 M Betaine, 6mM MgCl2, 1µM TSO (5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3') was added into the tube for RNA reverse transcription. The tubes containing total 10 µl mixture were put into ProFlex PCR System (Thermo Fisher Scientific) and the reaction condition was as follows: 42˚C for 90 min, followed by ten cycles of 50˚C for 2 min and 42˚C for 2 min. The final enzyme inactivation was performed at 70˚C for 15 min. After finishing the first-strand reaction, the 15µl PCR preamplification mix (1x KAPA HiFi HotStart ReadyMix, 0.1µM IS PCR primers (5'-AAGCAGTGGTATCAACGCAGAGT-3')) was added into the tube containing 10µl of the first-strand reaction. PCR preamplification reaction was performed in ProFlex PCR System by using the following program: 98˚C for 3 min, 12 cycles of 98˚C for 20s, 67˚C for 15s and 72˚C for 6 min, with the final extension cycle of 72˚C for 5 min. Amplified cDNA was purified by using Ampure XP beads (1:1 ratio) according to the manufacturer's manual and was eluted in 15 µl of ddH2O. Libraries were prepared using the Nextera XT kit and sequencing was carried out on an Illumina Hiseq 2000 using standard Illumina protocols.
attributes.tag: GEO Accession
attributes.value: GSM3672453