title: GSM1606361: ClutchB_polyA_21_hpf; Xenopus tropicalis; RNA-Seq
library_construction_protocol: For most samples, 10 embryos/timepoint were thoroughly homogenized in 200 ml Trizol® and frozen at -80oC, with the exception of egg, 0.5, 1, 1.5, 2, 2.5 and 3hpf timepoints, which were sampled at 25, 30, 20, 20, 20, 15 and 15 embryos, respectively. For the Clutch A timecourse, ERCC Spike In Mix 1 (10ul) (lot 1205006) was diluted in DEPC-treated water to a final volume of 1045ul. Diluted spike in mix was then pipetted directly into the Trizol homogenates such that 1ul was delivered per embryo, which in most cases required delivery of 10ul diluted spike in mix/sample, with the exception of the first several timepoints. Spike in mix was pipetted into samples in the order of their chronology. Total RNA was purified from the embryo homogenate according to the manufacturer's recommendations. Following isopropanol precipitation, RNAs were resuspended in DEPC-treated water and any contaminating genomic DNA was removed by a subsequent overnight precipitation in 5M LiCl at 4oC. RNA was pelleted and washed twice with 70% ethanol made with DEPC-treated water. All RNAs were resuspended and quantitated by Bioanalyzer. RNA quality was assessed by Bioanalyzer and RNA Integrity Numbers were all in excess of 9. For the Clutch B timecourse, ERCC spike in mix 1 (lot 1306008) was similarly diluted and delivered to all Trizol® homogenates, except the pipetting was done in reverse order to the chronology of the samples. Furthermore, we added an additional spike in mix that was pipetted independently and in order of sample chronology. Ambion ArrayControl™ spikes were used. Two microliters of spikes 1, 2 and 4 were diluted  to a final volume of 82ul in DEPC-treated water to create a solution of 2.439ng for each ArrayControl spike-in RNA per microliter. Approximately 5.125ul of this dilution was then further diluted into DEPC-treated water to a final volume of 1000ul as verified by weight to create a solution of ~12.5pg/ul. One microliter per embryo (~12.5pg) was added to Trizol® homogenates by pipetting of 10ul per tube except in the cases of the 0-3hpf samples. In both timecourses these early timepoint spike ins were pipetted by multiple rounds of 10ul (for 20 or 30ul delivery), or readjustment to 5ul (for 15ul delivery). RNAs were subsequently isolated as described above except we omitted the LiCl precipitation step. RNAs were similar quantitated and quality evaluated. RNA quality and concentration were evaluated by running total RNA on an Agilent RNA Nano Bioanalyzer chip. All samples had an RNA integrity number (RIN) greater than 9. Ribosomal RNA was removed via polyA-selection or ribosomal RNA depletion. For polyA-extraction, polyadenylated RNA transcripts were selected from the total RNA using two rounds of enrichment with oligo(dT)25 beads (Invitrogen). Ribosomal-depletion was completed with the Ribo-Zero (Human/Mouse/Rat) Magnetic Ribosomal rRNA Removal kit (Epicenter) according to the manufacturer's recommendations. The supernatant from both approaches was purified and concentrated using RNAclean XP beads (Beckman Coulter). Following selection, the RNA was fragmented to approximately 140 base pairs and first-strand synthesis was completed with SuperScript III reverse transcriptase (Invitrogen) using random hexamers. Magnetic AMPure XP beads (Beckman Coulter) were used to purify the cDNA and remained with the sample throughout sequencing library construction. During preparation, DNA is selectively precipitated by weight and re-bound to the beads through the addition of a 20% polyethylene glycol, 2.5 M NaCl solution. We used T4 DNA polymerase and T4 polynucleotide kinase to blunt ends and phosphorylate the fragments. The large Klenow fragment then added a single adenine residue to the 3' end of each fragment, custom adapters (IDT) are ligated using T4 DNA ligase, and, for strand-specific libraries, UDG removes uracil residues. The product is then PCR amplified using custom-made primers (IDT) containing unique 6 bp indices.
attributes.tag: GEO Accession
attributes.value: GSM1606361