Illuminatruseq stranded mrna sample prep kits

Normal HF-, Epd- and SCC-SCs were isolated as previously described (Ge et al., 2016 (link)). For wound-activated SCs (Wd-SCs), adult second telogen (P60) Sox9CreER; R26YFP female mice were subjected to full-thickness 7mm punch wounds. Seven days post-wounding, single cell suspensions were obtained by FACS as Lineage-YFP⁺ Integrin-α6⁺ (HF-derived) and Lineage-YFP^neg Integrin-α6⁺ (Epd-derived). Cells were lysed with TrizolLS (Invitrogen) and total RNA was isolated with the Direct-zol RNA MiniPrep kit (Zymo Research) and submitted to the Genomics Resources Core Facility of the Weill Cornell Medical College for quality control (determined using Agilent 2100 Bioanalyzer, with all samples passing the quality threshold of RNA integrity numbers (RIN 8). Library constructions were performed using IlluminaTruSeq Stranded mRNA Sample Prep Kits, and sequencing was performed on an Illumina HiSeq2000 sequencing machine.
For RNA quantitative PCR, primers DNA oligos were synthesized from Eurofinsgenomics complementary DNAs were generated from 1μg of total RNA using the SuperScript Vilo cDNA synthesis kit (Life Tech), diluted and used as templates for real-time PCR performed with the 7900HT Fast Real-Time PCR System (Applied Biosystems) and gene-specific primers listed in Table S2. Glyceraldehyde dehydrogenase (GAPDH) was used as a loading reference.

Prior to poly(A) RNA selection, 0.02 μl ERCC Spike-In Control Mix 1 (Life Technologies, Carlsbad, CA) was added to 1 μg total RNA. Poly(A)-selected stranded mRNA libraries were constructed from 1 μg total RNA using Illumina TruSeq Stranded mRNA Sample Prep Kits (Illumina, San Diego, CA) per the manufacturer’s instructions with the following exception: PCR amplification was performed for 10 cycles rather than 15 to minimize the risk of over-amplification. Unique barcode adapters were applied to each library; dual indices were used. Libraries were pooled for sequencing.
The pooled libraries were sequenced on multiple lanes of a HiSeq4000 using version 4 chemistry to achieve a minimum of 12 million 76-bp read pairs. The sequences were processed using RTA version 1.18.64 and CASAVA 1.8.2 (Illumina, San Diego, CA).