alias: GSM4544743
title: GSM4544743: Native young Rep1; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were isolated as described previously in (Habib et al., 2017). Per replicate, we isolated nuclei from the flash-frozen, whole hippocampus of the right hemisphere. All reagents were placed on ice. A lab dish was placed on ice and frozen tissue was placed on the dish before being covered with 1 ml lysis buffer from the Nuclei EZ Prep Kit (Sigma-Aldrich, St. Louis, USA). Tissue was cut into 2 mm-small pieces and the tissue-buffer mix was transferred on a 2 ml tissue grinder placed on ice. 1 ml of fresh lysis buffer was used to rinse the lab dish and then loaded on the tissue grinder as well (2 ml total volume). Tissues were homogenized with 25 strokes with pastel A followed by 15 strokes with pastel B. Tissue homogenate was transferred to a fresh tube on ice, and the tissue grinder was rinsed with 2 ml of fresh lysis buffer, which were subsequently added to said tube (4 ml total volume), followed by 5 min incubation on ice. Nuclei were pelleted with 5 min centrifugation at 500g, before resuspending the pellet with 4 ml fresh lysis buffer. Following further 5 min centrifugation at 500g, lysis buffer was removed and the pellet washed with 4 ml PBS. Nuclei were pelleted and resuspended in 500 μl PBS containing 2 U/ml Protector RNase Inhibitor (Sigma-Aldrich, St. Louis, USA). Nuclei solution was parsed through a 40 μm mesh. Nuclei were counted and concentrations adjusted to ~1 mio nuclei/ml. Reagents of the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10X Genomics, Pleasanton, USA) were thawed and prepared according to the manufacturer's protocol. Nuclei/master mix solution was adjusted to target 7,500 nuclei per sample and loaded on a standard Chromium Controller (10X Genomics, Pleasanton, USA), according to the manufacturer's protocol. All reaction and quality control steps, including library construction (using Chromium Single Cell 3′ Library Construction Kit v3) were conducted according to the manufacturer's protocol and with recommended reagents, consumables and instruments. Quality control of cDNA and libraries was conducted using a Bioanalyzer (Agilent, Santa Clara USA) at the Stanford Protein and Nucleic Acid Facility (http://pan.stanford.edu/index.html).    Native libraries: Illumina sequencing of 10X snRNA-seq libraries was performed by Novogene Co. Inc. (Sacramento, US; https://en.novogene.com/). Multiplexed libraries were sequenced with 2x150-bp paired-end (PE) reads in a single S4 lane on a Illumina Novaseq S4 (Illumina, San Diego, USA), targeting 215 million reads per library. Base calling, demultiplexing and generation of FastQ files was conducted by Novogene.    Converted libraries: Libraries generated using the Chromium 10X Single Cell 3' (version 3) kit require a conversion step using the MGIEasy Universal Library Conversion kit (Part Number: 1000004155, MGI Tech Co., Ltd, Shenzhen, China) before DNA nanoballs (DNBs) can be generated. For each library, 50 ng of final 10X library product was further amplified using 5 cycles of PCR to incorporate a 5' phosphorylation on the forward strand only. Purified PCR product was then denatured and mixed with a “splint” oligonucleotide that is homologous to both P5 and P7 adapter regions of the library to generate a circle. A ligase reaction was then performed to create a complete ssDNA circle of the forward strand. Then an exonuclease digest was performed to remove single stranded non-circularized DNA molecules and splint oligonucleotide. DNBs were then prepared using the MGI CoolMPS PE100 beta sets now available as commercial products (Part Number 1000016935, MGI Tech Co., Ltd, Shenzhen, China). Rolling circle replication time for DNB preparation was set to 25 min. DNBs were loaded onto DNBSEQ-G400 4-lane flowcell provided with the PE100 sequencing set by manual pipetting of DNBs mixed with loading buffer into flowcell lanes using the MGIDL-200H DNB loader as described in the CoolMPS High-throughput Sequencing Set User Manual provided with the kit. Sequencing primers in the MGI CoolMPS PE100 kit were replaced with the primers provided in the High-Throughput Pair-End Sequencing Primer Kit (App-A) (Part Number 1000004156, MGI Tech Co., Ltd, Shenzhen, China) according to the instructions described in the Primer Kit manual. Sequencing was performed with a customized program with 28 sequencing cycles (cell barcode) for the 1st strand and 98 cycles of cDNA followed by 8 cycles of the sample barcode reading for the 2nd strand.  Base calling and generation of FastQ files on the DNBSEQ-G400 was performed using the software release for CoolMPS (BasecallLite version_1.0.7.84).
identifiers.namespace: GEO
identifiers.id: GSM4544743