GSM9523460: ESC2i Bod1L WT biol rep 2; Mus musculus; RNA-Seq
Total RNA was isolated with Trizol (Invitrogen) according to the manufacturer's recommendations. 100 μg total RNA was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit; QIAGEN), followed by DNaseI treatment (QIAGEN). 100–200 ng mRNA was fragmented by hydrolysis (5× fragmentation buffer: 200mM Tris acetate, pH8.2, 500mM potassium acetate and 150mM magnesium acetate) at 94°C for 90 s and purified (RNAeasy Minelute Kit; QIAGEN). cDNA was synthesized with 5 μg random hexamers by Superscript III Reverse Transcriptase (Invitrogen). Ds cDNA synthesis was performed in second strand buffer (Invitrogen) according to the manufacturer's recommendations and purified (Minelute Reaction Cleanup Kit; QIAGEN). Strand-specific rRNA depleted ds cDNA profiling was performed with the ScriptSeq kit (cat. no. SS10924) from Illumina, according to the instructions of the manufacturer. rRNA depletion was performed with the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat; cat. no. RZH110424).