alias: GSM3228167
title: GSM3228167: MM45_E2.5_Mettl16_WT; Mus musculus; RNA-Seq
library_construction_protocol: For single-embryo transcriptome sequencing, the isolated E2.5 and E3.5 embryos were visually examined for viability and cell number, and transferred separately into single tubes of 0.2 ml thin-walled 8-tube PCR strips (Thermo, AB-0451). The tubes contained 2 μl of the following mix: 0.4% Triton-X100 (vol/vol) in H2O + 2U/μl SUPERase• In™ RNase Inhibitor (20 U/μL; Thermo, AM2694). Embryos were stored at -80°C prior to processing for Smart-seq2 library preparation.   For in vitro methylation with METTL16 the libraries of randomized 30 nt RNA sequences were chemically synthesized. The sequences had a constant central 9-mer sequence flanked by randomized (represented by N) sequences (MM-RNA-14: N11-UACAGAGAA-N10). In vitro methylation with METTL16 protein was followed by m6A immunoprecipitation using rabbit polyclonal antibody (5 µg; Synaptic Systems, catalog no. 202003). Polyadenylated transcripts from single embryos (E2.5 or E3.5) were amplified using the Smart-seq2 protocol. The protocol generates libraries that lack strand specificity. Multiple libraries with different barcodes were mixed in equimolar ratios and paired-end sequencing reads were obtained with the NextSeq 500 (EMBL GeneCore facility, Heidelberg). The maximum sequencing length was 80 nt.   Sequencing libraries  for in vitro methylation experiments were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Cat No:E7300) according to manufacturer instructions and sequenced on Illumina HiSeq 2000 platform (EMBL Heidelberg Gene core facility).
identifiers.namespace: GEO
identifiers.id: GSM3228167