title: GSM1486498: ES_Tsix-stop T=2d; Mus musculus; RNA-Seq
library_construction_protocol: 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 (5x 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 using 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 used for the NPC lines 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 using 5 μg of total RNA (Human/Mouse/Rat; cat. no. RZH110424). Collection of the cells for Hi-C and the HiC sample preparation procedure was performed as previously described (Lieberman-Aiden et al., 2009), with the slight modification that DpnII was used as restriction enzyme during initial digestion. For the PolyA+ samples, cDNA was prepared for sequencing by end repair of 20 ng ds cDNA as measured by Qubit (Invitrogen). Adaptors were ligated to DNA fragments, followed by size selection (~300 bp) and 14 cycles of PCR amplification. Quality control of the adaptor-containing DNA libraries of both PolyA+ and ScriptSeq samples was performed by qPCR and by running the products on a Bioanalyzer (BioRad). Cluster generation and sequencing (32-42 bp) was performed with the Illumina Genome Analyzer IIx or Hi-Seq 2000 platforms according to standard Illumina protocols. For HiC, paired-end libraries were prepared according to Lieberman-Aiden et al. (2009) and sequenced on the NextSeq 500 platform using 2*75 bp sequencing.
attributes.tag: GEO Accession
attributes.value: GSM1486498