title: GSM1430929: RNAseq_single_embryo_replicate4; Xenopus laevis; RNA-Seq
library_construction_protocol: Total RNA was isolated using TRizol® Reagent (Ambion) and RNeasy Kit (Quiagen), and rRNA depleted with Ribo-Zero™ rRNA Removal Kits (Epicentre). rRNA-depleted RNA was then heat-fragmented in 40mMTris-acetate, 100mM potassium acetate and 30mM magnesium acetate (pH8.2) and used for cDNA synthesis. First cDNA strand was obtained from random examers primers using Superscript III (Invitrogen); second strand was made with E.coli DNA polymerase I (NEB), E.coli DNA ligase (NEB) and T4 DNA polymerase (Promega). Purified cDNA was used for strand-specific Illumina sample preparation. Libraries were constructed according to standard Illumina protocols. Briefly cDNA was ligated with adapter sequences, size selected (300bp), and amplified by PCR. For the embryo samples purified cDNA was used for strand-specific Illumina sample preparation using Kapa Hyper Prep Kit, according to manufacturer protocol. To confer strand-specificity a USER enzyme (NEB) digestion step was performed prior to library amplification.