alias: GSM3473725
title: GSM3473725: Wnt-LOF_st9p_polyA-RNA_rep2; Xenopus tropicalis; RNA-Seq
library_construction_protocol: Total RNA extraction: 10-15 dejellied X. tropicalis embryos were homogenized in 800 µl TRIzol reagent by vortexing. For phase separation, the homogenate supplemented with 1/5 volume of chloroform was transferred to a 1.5-ml phase-lock gel heavy microcentrifuge tube, shaken vigorously for 15 sec, left on the bench for 2 min and centrifuged (16,000 g) for 5 min at 4ºC. The upper phase was mixed with 1 volume of absolute ethanol and centrifuged (12,000 g) through the column IIC of Zymo RNA Clean and Concentrator-25 (Zymo Research) for 30 sec at room temperature. Next, the manufacturer's instructions were followed for the recovery of total RNA (>17 nt) with minor modifications. First, the flow-through of the first spin was re-applied to the column. Second, the RNA was treated in-column with Turbo DNase (2 U/µl, ThermoFisher Scientific). Third, the RNA was eluted twice with 25 µl molecular-grade water. The concentration was determined on the Nanodrop 1000 except for RNA samples subjected to ribosomal RNA depletion in which case more accurate fluorometry was used. Metabolic labeling of nascent transcripts: 75 ng 4-thiouridine-5'-triphosphate (4sU) was injected into the zygote's animal pole. Total RNA was extracted as described above from 30 embryos collected at the mid-blastula transition (stage 8+) and early gastrula stage (stage 10+). Ribosomal RNA (rRNA) was removed from ~10 µg total RNA with Ribo-Zero rRNA (human/mouse/rat) magnetic beads (Illumina) following the manufacturer's instructions. rRNA-depleted total RNA was purified with the RNA Clean & Concentrator-5 kit (Zymo Research) according to the manufacturer's instructions. The yield was determined on a fluorometer using high-sensitivity reagents for RNA (250 pg/µl to 100 ng/µl). The purified RNA was transferred to thin-walled PCR tubes and fragmented with 40 µl 1x NEBNext RNA fragmentation buffer (NEB) in a thermocycler for 4 min at 95ºC. The fragmentation was stopped on ice with NEBNext RNA fragmentation stop solution (NEB) before purifying the fragmented RNA with RNA Clean & Concentrator-5. The RNA was eluted twice with 15 µl molecular-grade water. Approximately 10 ng of RNA was set apart to make an input library. The remaining RNA was biotinylated in the dark with EZ-Link HPDP-Biotin for 3 h at room temperature. The RNA was purified with RNA Clean & Concentrator-5 and eluted twice with 20 µl molecular-grade water. Biotinylated RNA was separated from non-labelled RNA with M270 streptavidin magnetic beads following the manufacturer's instructions. The biotinylated RNA was stripped off the beads twice with 100 µl 100 mM 2-mercaptoethanol (pre-heated to 80ºC) and purified with RNA Clean & Concentrator-5. RNA-Seq: Indexed paired-end libraries were made from ~1 µg total RNA by following the low sample protocol of the TruSeq RNA sample preparation guide v2 with a few modifications. First, cDNA after second strand synthesis was quantified using a fluorometer and high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl). Second, the number of PCR cycles was adjusted to the detected yield of cDNA to avoid products of over-amplification such as chimera fragments: 7 (~20 ng), 8 (~10 ng), 9 (~5 ng), 12 (~1 ng). 4sU-Seq: Input and biotinylated RNA were converted to indexed paired-end libraries using the ScriptSeq v2 RNA-Seq preparation kit (Epicentre). We used the manufacturer's instructions (protocol rev. C, 8/2014) starting with appendix 4.1.A (annealing cDNA synthesis primer) and 4.1.B (cDNA synthesis) followed by main protocol section 3.C (synthesis of 3'-tagged DNA). The DNA libraries were purified using 1.8x Agencourt AMPure XP beads and amplified with 11 (input) and 15 (streptavidin pull-down) PCR cycles according to section 3.D to 3.F. The DNA from the PCR reaction was purified using 1x AMPure XP beads and eluted in 10 µl molecular-grade water. Illumina libraries were sequenced paired-end on the HiSeq 2500 (4sU-Seq) and HiSeq 4000 (RNA-Seq). RNA-Seq: Indexed paired-end libraries were made from ~1 µg total RNA by following the low sample protocol of the TruSeq RNA sample preparation guide v2 with a few modifications. First, cDNA after second strand synthesis was quantified using a fluorometer
identifiers.namespace: GEO
identifiers.id: GSM3473725