TITLE: Library Generation using Slide-seqV2
AUTHORS: Robert   Stickels, Evan Murray, Jamie Marshall, Karol Balderrama, Irving Barrera, Evan Macosko, Fei Chen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol detailing the steps necessary to generate libraries using previously manufactured Slide-seq arrays. </div></div>

[STEPS]
?. [Tissue Sectioning and RNA capture]
Equilibrate fresh frozen tissue to -20 °C in a cryostat for ~20 minutes prior to sectioning. The tissue can be mounted onto a chuck using OCT, and the tissue should be aligned and sectioned as is standard in other protocols. Sections for Slide-seq should be 10 μm in thickness.
?. [Tissue Sectioning and RNA capture]
Carefully with thin forceps, place a Slide-seq array (puck) on a glass slide and set it in the cryostat to cool. Once cold, a tissue section should be moved onto the puck using a brush. Take care to position the target region of interest over the puck if the tissue section is larger than the puck.
?. [Tissue Sectioning and RNA capture]
Once the tissue is positioned correctly, lift the glass slide and place a finger on the slide underneath where the puck and tissue are located. As the region warms, the tissue will melt onto the puck. To avoid curling of the tissue, it's best to start from one side and slowly move your finger across the region rather than warming it from the center.
?. [Tissue Sectioning and RNA capture]
Using forceps, move the puck into a 1.5 mL tube containing 200 μL of hybridization buffer. Note that from this point forward, all steps must be performed using Eppendorf DNA Lo-bind tubes or equivalent in order to prevent beads from sticking to the sides of the tubes.
?. [Tissue Sectioning and RNA capture]
Hybridization Buffer per puck:    190 μL 6x SSC    10 μL RNase InhibitorTotal volume 200 μL
?. [Library Preparation]
Incubate each puck in the hybridization buffer for 15 minutes at room temperature. During this time, prepare the reverse transcription mix and additional 1x Maxima RT buffer. It is possible to leave the pucks in the hybridization buffer for up to 30 minutes without adversely affecting the quality of the data.
?. [Library Preparation]
Using forceps, carefully remove each puck from the hybridization buffer and dip it gently into a tube of 1x Maxima RT buffer to equilibrate the puck to that buffer. After this dip, move it to a new tube containing 200 μL of reverse transcription mix. Allow pucks to incubate in the reverse transcription mix first for 30 minutes at room temperature followed by an additional incubation for 90 minutes at 52 °C. The total incubation time is 2 hours.
?. [Library Preparation]
Reverse Transcription Mix per puck:    115 μL Ultrapure water    40 μL Maxima 5x RT Buffer    20 μL 10 mM dNTPs     5 μL RNase Inhibitor    10 μL 50 uM Template Switch Oligo    10 μL Maxima H- RTase EnzyneTotal volume 200 μL
?. [Library Preparation]
Add proteinase K to the tissue clearing buffer stock solution at a ratio of 1:50 to create a working solution for tissue digestion. Add 200 μL of this working solution to each tube and pipet gently to mix within the tube without disrupting the puck. Incubate for 30 minutes at 37 °C. The total volume in each tube should be 400 μL at this point.
?. [Library Preparation]
Tissue Clearing Buffer Stock Recipe:    100 mM Tris-HCl pH 7.5    200 mM NaCl    2% SDS (w/v)    5 mM EDTAWorking solution for Step 7 per puck:    196 μL Tissue clearing buffer stock    4 μL Proteinase K enzymeThe tissue clearing buffer (without proteinase K) can be made ahead of time and stored for up to several months at room temperature.
?. [Library Preparation]
Add 200 μL TE-TW to each tube and pipet several times to mechanical shear any tissue remaining in the tube or on the puck. At this time, the individual beads should also release from the coated glass piece which held the puck. Taking care not to introduce any bubbles, pipet several times until all beads have visibly been removed. Using forceps, quickly remove the glass shard in the tube before the beads start to pellet. At this point, ensure all beads are still suspended in the mixture and transfer all volume to a new Lo-bind tube. The total volume per tube should be 600 μL at this step.
?. [Library Preparation]
Centrifuge each sample for 2 minutes at 3000 RCF. A white pellet should be clearly visible. Remove the supernatant and resuspend the beads carefully in 200 μL TE-TW. It is best practice not to allow any beads to entire the pipet tip from this point forward so as to minimize any bead loss that might result from sticking to the sides of the tip. Perform this TE-TW wash a total of 2 times at this step. It is possible to pause here for up to 3 days. Store beads in 200 μL TE-TW at 4 °C after washes are completed.
?. [Library Preparation]
Pellet beads and resuspend in 10 mM Tris-HCl, pH 7.5. Pellet once again and resuspend in 200 μL Exonuclease I mix. Incubate at 37 °C for 50 minutes.
?. [Library Preparation]
Exonuclease I mix per puck:    170 μL ultrapure water    20 μL 10x exonuclease I buffer    10 μL exonuclease I enzymeTotal volume 200 μL
?. [Library Preparation]
Add 200 μL TE-TW to each sample and pellet as before. Wash each sample a total of 2 times with TE-TW. Pellet the beads once more and resuspend in 200 μL 0.1 N NaOH and incubate for 5 minutes at room temperature. After the incubation, add 200 μL TE-TW to quench the NaOH. Note: 0.1 N NaOH should be prepared immediately prior to each use.
?. [Library Preparation]
Pelleting as before, wash the beads once with TE-TW and then once again with 1x TE buffer (no tween added). Finally, pellet the beads once more and resuspend in 200 μL second strand synthesis mix. Incubate for 1 hour at 37 °C.
?. [Library Preparation]
Second strand synthesis mix per puck:    133 μL ultrapure water    40 μL Maxima 5x RT Buffer    20 μL 10 mM dNTPs    2 μL 1 mM dN-SMRT oligo    5 μL Klenow EnzymeTotal volume 200 μL
?. [Library Preparation]
Add 200 μL TE-TW to each sample and then transfer all beads to new Lo-bind tubes. As before, pellet the beads and resuspend in TE-TW a total of 3 times. Pellet the beads once more and then resuspend in 200 μL ultrapure water. Finally, pellet once more and resuspend in 200 μL PCR mix. Note: beads will not pellet as readily in water compared to the washes. It is critical to ensure that all beads have pelleted before removing the supernatant at this step. It helps to rotate the tubes in the centrifuge and perform an additional centrifugation step.
?. [Library Preparation]
PCR mix per puck:    88 μL ultrapure water    100 μL Terra PCR Direct Buffer    4 μL 100 μM Truseq PCR primer     4 μL 100 μM SMART PCR primer     4 μL Terra Polymerase  Total volume 200 μL
?. [Library Preparation]
After the beads are resuspended in the PCR mix, we find it works best to divide the total volume of each sample into 4 PCR tubes each containing 50 μL (25%) of the total. Check that the beads have not pelleted inside the PCR tubes and then run the following PCR protocol:Slide-seq WTAStart:    98 °C, 2 minutes 4 cycles of:     98 °C, 20 seconds     65 °C, 45 seconds     72 °C, 3 minutes 9 cycles of:     98 °C, 20 seconds     67 °C, 20 seconds     72 °C, 3 minutes Then:     72 °C, 5 minutes    4 °C, Hold It is possible to pause here. Store the samples at 4 °C.
?. [Purification and Quantification]
Recombine the samples that were split into 4 parts in the previous step and perform a PCR clean-up using AMPure XP beads following the manufacturer's guidelines. In particular, we use a ratio of 0.6x AMPure beads (120 μL AMPure beads into 200 μL sample volume) for all steps. We perform the purification twice, eluting first in 50 μL water, proceeding again with a ratio of 0.6x (30 μL AMPure beads into 50 μL sample volume), and finally eluting into 20 μL water to obtain the final product.
?. [Purification and Quantification]
To quantify the cDNA libraries, we use both the Qubit dsDNA high sensitivity kit and Bioanalyzer High Sensitivity DNA kit following the manufacturer protocols. As a general guideline, little variability is expected between replicates within a single tissue sample, but larger variability may be observed between tissues. Concentrations in the range of approximately 0.3 ng/μL and above are acceptable, and expected average fragment sizes fall roughly within the range of 1300-1700 base pairs when following this protocol. There should be no significant amount of primer dimer present.
?. [Tagmentation]
The tagmentation is performed using the Nextera XT DNA Library prep kit largely following the manufacturer protocols. However, some modifications are necessary and are outlined in a brief protocol as follows:
?. [Tagmentation]
Pre-heat a thermocycler to 55 °C. While the block is heating, prepare 600 picogram dilutions of all samples into a total volume of 5 μL of ultrapure water. Add tagmentation buffer to each sample and then add the transposase enzyme immediately before moving to 55 °C. Incubate for 5 minutes. Note: steps in the tagmentation protocol are highly time-sensitive. Use of a multi-channel pipet is recommended to hasten pipetting steps and facilitate rapid mixing at each step.
?. [Tagmentation]
Tagmentation Mix per sample:    600 pg cDNA in total volume of 5 μL ultrapure water    10 μL Tagment DNA Buffer    5 μL Amplicon Tagment EnzymeTotal volume 20 μL
?. [Tagmentation]
Add 5 μL of NT buffer as quickly as possible following the tagmentation reaction. Mix quickly using a multi-channel pipet and allow to incubate at room temperature for 5 minutes.
?. [Tagmentation]
Prepare the Nextera PCR Master mix with water, NPM, and P5-Truseq Hybrid oligo. Add 24 μL of this master mix to each sample, followed by 1 μL of i7 indexing primer from the Nextera XT kit. Gently mix by pipetting and run the PCR program below.Note: Each sample must use a different i7 index if you intend to pool samples for multiplexed sequencing. We do not recommend dual-indexing of samples. It is possible to pause here. Store the samples at 4 °C after the PCR.
?. [Tagmentation]
Nextera PCR Mix per sample:    8 μL ultrapure water    15 μL NPM    1 μL of 10 μM P5-Truseq Hybrid oligo    1 μL of Nextera N7XX indexing primer or custom i7 index    25 μL from tagmentation reactionTotal volume 50 μL
?. [Tagmentation]
Nextera XT PCR Amplification ProtocolStart    72 °C, 3 minutes     95 °C, 30 seconds 12 cycles of:     95 °C, 10 seconds     55 °C, 30 seconds     72 °C, 30 seconds Then:     72 °C, 5 minutes    4 °C, Hold
?. [Tagmentation]
Purification and Quantification of DNA sequencing libraries is performed similarly to the cDNA purification and quantification in steps 15 and 16. However, it is only necessary to perform the AMPure clean-up once. Additionally, final product should be eluted in 10 μL. Variability between samples is common. As a general guideline, acceptable concentrations for libraries are approximately 3 ng/μL or higher and expected average fragment sizes are in the range 400-700 base pairs.
?. [Sequencing]
Sequencing is standard following Illumina's protocols, and submission should proceed as outlined by your institution's core facility or lab policies. We recommend preparing pools of libraries at 4 nM concentration if possible. For best results, it's generally advised to sequence each puck to a depth of 100-200 million reads, though smaller sequencing runs can be used to assess the quality of new samples. Read structure is as follows:Read 1: 42 bpIndex 1: 8 bpRead 2: 41 - 60 bp Index 2: 0 bpFor read 2, the read length is dependent on the capacity of the sequencing kit. For a Nextseq 75-cycle kit, the maximum possible is 41 bp, which is the minimum recommended length. For a 100-cycle kit, use a read length of 60 bp.