title: GSM3732301: MFI131; Macaca fascicularis; RNA-Seq
library_construction_protocol: Dissect the implantation site of the embryos on Matrigel and remove the extra-embryonic cells under stereo microscope remaining the bi-laminar disc-like structure visualized clearly. Put the embryonic fragment in drops with 1 X TrypLE for 15 min at 37°C and pipette the digested tissue into single cells by repeated mouth pipetting. Then collect the cells into 500 μl CRML 1066 containing 10% FBS to terminate the enzymatic reaction and centrifuge to collect cells at 300 g for 5 min at room temperature. Resuspend the cell pellet with 0.1% BSA/PBS and store at 4°C. Pick single cells into 2.5 µl lysis buffer in 200 µl PCR tubes. The linearized RNA molecules were released in a thermocycler at 72 °C for 3 min at first. Then, transfer the reactions immediately on ice. Add 2.85 µl RT mixture into every reaction with component as SuperScript II reverse transcriptase, RNase Inhibitor, 5× Superscript II first-strand buffer, dithiothreitol (DTT), betaine, MgCl2, and template switch oligo (TSO) primer. Spin down the 5.4 µl samples in the thermocycler at 25 °C for 5 min, 42 °C for 60 min, 50 °C for 30 min, and then 70 °C for 10 min, 4 °C hold. Prepare 7.5 µl PCR mixture (6.25 μL 2× KAPA HiFi HotStart ReadyMix, ISPCR oligo, and 3′ Anchored oligo) to add in every tube reaction, and amplify samples for 19 cycles. Pool together samples with different barcodes (up to 96 barcodes) and purify DNA. Then prepare the second round of amplification with biotinylated index primer and ISPCR oligo for an additional five cycles. Subsequently, purify the biotinylated cDNAs further with 0.8× Ampure XP beads and elute cDNA in 50 µl EB. Next, shear the cDNAs into approximately 300-bp fragments with sonicator. Enrich the cDNA fragments by Dynabeads® MyOneTM Streptavidin C1. Libraries were prepared with KAPA Hyper Prep Kits. Use the NEB U-shaped adapter for ligation. Finally, amplify the adapter-ligated fragments using an Illumina QP2 primer and a short universal primer for 9 cycles.
attributes.tag: GEO Accession
attributes.value: GSM3732301