GSM2064368: RBPjk null_sample2; Mus musculus; RNA-Seq
Skin samples were embedded in OCT immediately after collecting and snap-frozen in liquid nitrogen before storing at -80°C Sectioned at 20μm thickness, tissue slices were placed on poly-lysine (Sigma-P8920; 1:10 dilution) treated PEN membrane glass slides (Arcturus, LCM0522), dehydrated in EtOH, and equilibrated to Xylene before the LCM procedure Laser capture was performed with Arcturus Veritas and Arcturus CapSure HS LCM Caps (Arcturus Engineering, Inc., Mountain View, CA) within 45 minutes to avoid degradation. 4 membrane with ~25-30 hair follicles each were pooled and stored in QIAzol (Qiagen) at -80°C. RNA extracted as described in methods was QCed for sequencing using an Agilent 2100 Bioanalyzer requiring integrity numbers >8 to pass. Libraries were prepared with the Nextera XT DNA Sample Preparation kit (Illumina Technologies). 1ng of cDNA was tagmented with the Amplicon Tagment Mix at 55°C for 10min. NT Buffer was added to neutralize the samples. Libraries were PCR amplified (Nextera PCR Master Mix) in the following program: one cycle of 72°C for 3min, one cycle of 98°C for 30s, 12 cycles of 95°C for 10s, 55°C for 30s, and 72°C for 1min, and one cycle of 72°C for 5min and (N7XX, and N5XX). The purified cDNA was captured on an Illumina flow cell for cluster generation and sequenced (75bp paired-end, 20 million reads, Illumina HiSeq2500) following the manufacturer's protocol.
GEO Accession
GSM2064368