Name: GSM7183980
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: OTHER
Selection: other
Layout: SINGLE
Construction protocol: ~200 oocytes or zygote, or ~100 early 2-cell embryos were used as one group for low input ribo-seq. Collected samples were washed with PBS and transferred into 1.5 ml nuclease free centrifuge tubes with a minimal volume of PBS. The tubes were frozen immediately in dry ice and stored at -80 until use. The EZ-Manga RIP kit (Millipore 17-701) and anti-HA beads (Pierce 88836) were used for the low input ribo-seq experiment. Samples were first lysed following the RIP kit manual and then 100U RNase I (Ambion AM2294) was added to each tube for RNA digestion. The digestion was done at room temperature for 45 min. After digestion, anti-HA magnetic beads were added to each tube to finish IP at 4 degrees for 4h following the manual. 7ul ultrapure water supplied with 0.25 ul RNase inhibitor from the SMARTer smRNA-Seq Kit (Takara 635029) was added to each tube and the tubes were heated for 15min at 70 degrees to denature the proteins and RNAs inside. The tubes were transferred to ice immediately after heating and the remaining part of the manual was followed to finish library construction. 22 PCR cycles were used for indexing of libraries and the finial PCR products were purified with AMPure XP beads (Bechman A63881) for 2 time at 1:1.5 ratio.